Novel cas13b orthologues crispr enzymes and systems

ABSTRACT

The invention provides for systems, methods, and compositions for targeting nucleic acids. In particular, the invention provides non-naturally occurring or engineered RNA-targeting systems comprising a novel RNA-targeting Cas13b effector protein and at least one targeting nucleic acid component like a guide RNA or crRNA.

RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

This application is a continuation application of U.S. patentApplication Ser. No. 16/493,464, feed Sep. 12, 2019, which is a U.S.National Stage application of International Application No.PCT/US2018/022751, filed Mar. 15, 2018 which claims the benefit of toU.S. provisional application 62/471,710, filed Mar. 15, 2017 and U.S.provisional application 62/566,829, filed Oct. 2, 2017, the contents ofwhich are hereby incorporated in their entireties. Reference is made toPCT application including as it designates the U.S., inter alia,application No. PCT/US2016/058302, filed Oct. 21, 2016. Reference ismade to U.S. provisional patent application 62/245,270 filed on Oct. 22,2015, U.S. provisional patent application 62/296,548 filed on Feb. 17,2016, and U.S. provisional patent applications 62/376,367 and62/376,382, filed on Aug. 17, 2016. Reference is further made to U.S.provisional 62/471,792, filed Mar. 15, 2017 and U.S. provisional62/484,786, filed Apr. 12, 2017. Mention is made of: Smargon et al.(2017), “Cas13b Is a Type VI-B CRISPR-Associated RNA-Guided RNaseDifferentially Regulated by Accessory Proteins Csx27 and Csx28,”Molecular Cell 65, 618-630 (Feb. 16, 2017) doi:10.1016/j.molcel.2016.12.023. Epub Jan. 5, 2017 and Smargon et al.(2017), “Cas13b Is a Type VI-B CRISPR-Associated RNA-Guided RNaseDifferentially Regulated by Accessory Proteins Csx27 and Csx28,” bioRxiv092577; doi: https://doi.org/10.1101/092577. Posted Dec. 9, 2017. Eachof the foregoing applications and literature citations are herebyincorporated herein by reference.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grant numbersMH100706 and MH110049 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

SEQUENCE LISTING

The contents of the electronic sequence listing (“BROD-2780US-CONST26.xml”; Size is 791,332 bytes and it was created on Jul. 10, 2023) isherein incorporated by reference in its entirety.

Indeed, all documents cited or referenced herein and in herein citeddocuments, together with any manufacturer's instructions, descriptions,product specifications, and product sheets for any products mentionedherein or in any document incorporated by reference herein, are herebyincorporated herein by reference, and may be employed in the practice ofthe invention. More specifically, all referenced documents areincorporated by reference to the same extent as if each individualdocument was specifically and individually indicated to be incorporatedby reference.

FIELD OF THE INVENTION

The present invention generally relates to systems, methods andcompositions used for the control of gene expression involving sequencetargeting, such as perturbation of gene transcripts or nucleic acidediting, that may use vector systems related to Clustered RegularlyInterspaced Short Palindromic Repeats (CRISPR) and components thereof.

BACKGROUND OF THE INVENTION

The CRISPR-CRISPR associated (Cas) systems of bacterial and archaealadaptive immunity are some such systems that show extreme diversity ofprotein composition and genomic loci architecture. The CRISPR-Cas systemloci has more than 50 gene families and there is no strictly universalgenes indicating fast evolution and extreme diversity of lociarchitecture. So far, adopting a multi-pronged approach, there iscomprehensive cas gene identification of about 395 profiles for 93 Casproteins. Classification includes signature gene profiles plussignatures of locus architecture. A new classification of CRISPR-Cassystems is proposed in which these systems are broadly divided into twoclasses, Class 1 with multisubunit effector complexes and Class 2 withsingle-subunit effector modules exemplified by the Cas9 protein. Noveleffector proteins associated with Class 2 CRISPR-Cas systems may bedeveloped as powerful genome engineering tools and the prediction ofputative novel effector proteins and their engineering and optimizationis important. Novel Cas13b orthologues and uses thereof are desirable.

Citation or identification of any document in this application is not anadmission that such document is available as prior art to the presentinvention.

SUMMARY OF THE INVENTION

Effector proteins include two subgroups, Type VI-B 1 and Type VI-B2, andinclude members which are RNA-programmable nucleases, RNA-interferingand may be involved in bacterial adoptive immunity against RNA phages. ACas13b system can comprise a large single effector (approximately 1100amino acids in length), and one or none of two small putative accessoryproteins (approximately 200 amino acids in length) nearby a CRISPRarray. Based on the nearby small protein, the system is bifurcated intotwo Loci A and B. No additional proteins out to 25 kilobase pairsupstream or downstream from the array are conserved across species witheach locus. With minor exceptions, the CRISPR array comprises directrepeat sequences 36 nucleotides in length and spacer sequences 30nucleotides in length. The direct repeat is generally well conserved,especially at the ends, with a GTTG/GUUG at the 5′ end reversecomplementary to a CAAC at the 3′ end. This conservation suggests strongbase pairing for an RNA loop structure that potentially interacts withthe protein(s) in the locus. A motif search complementary to the directrepeats revealed no candidate tracrRNAs nearby the arrays, possiblyindicative of a single crRNA like that found in the Cpf1 locus.

In embodiments of the invention, a Type VI-B system comprises a novelCas13b effector protein and optionally a small accessory protein encodedupstream or downstream of the Cas13b effector protein. In certainembodiments, the small accessory protein enhances the Cas13b effector'sability to target RNA.

The invention provides a non-naturally occurring or engineeredcomposition comprising

-   -   i) a certain novel Cas13b effector protein, and    -   ii) a crRNA,    -   wherein the crRNA comprises a) a guide sequence that is capable        of hybridizing to a target RNA sequence, and b) a direct repeat        sequence,    -   whereby there is formed a CRISPR complex comprising the Cas13b        effector protein complexed with the guide sequence that is        hybridized to the target RNA sequence. The complex can be formed        in vitro or ex vivo and introduced into a cell or contacted with        RNA; or can be formed in vivo.

In some embodiments, a non-naturally occurring or engineered compositionof the invention may comprise an accessory protein that enhances TypeVI-B CRISPR-Cas effector protein activity.

In certain such embodiments, the accessory protein that enhances Cas13beffector protein activity is a csx28 protein. In such embodiments, theType VI-B CRISPR-Cas effector protein and the Type VI-B CRISPR-Casaccessory protein may be from the same source or from a differentsource.

In some embodiments, a non-naturally occurring or engineered compositionof the invention comprises an accessory protein that represses Cas13beffector protein activity.

In certain such embodiments, the accessory protein that represses Cas13beffector protein activity is a csx27 protein. In such embodiments, theType VI-B CRISPR-Cas effector protein and the Type VI-B CRISPR-Casaccessory protein may be from the same source or from a differentsource. In certain embodiments of the invention, the Type VI-BCRISPR-Cas effector protein is from Table 1A or 1B. In certainembodiments, the Type VI-B CRISPR-Cas accessory protein is from Table 1Aor Table 1B.

In some embodiments, a non-naturally occurring or engineered compositionof the invention comprises two or more crRNAs.

In some embodiments, a non-naturally occurring or engineered compositionof the invention comprises a guide sequence that hybridizes to a targetRNA sequence in a prokaryotic cell.

In some embodiments, a non-naturally occurring or engineered compositionof the invention comprises a guide sequence that hybridizes to a targetRNA sequence in a eukaryotic cell.

In some embodiment, the Cas13b effector protein comprises one or morenuclear localization signals (NLSs).

The Cas13b effector protein of the invention is, or in, or comprises, orconsists essentially of, or consists of, or involves or relates to sucha protein from or as set forth in Table 1A or Table 1B. This inventionis intended to provide, or relate to, or involve, or comprise, orconsist essentially of, or consist of, a protein from or as set forth inTable 1A or Table 1B, including mutations or alterations thereof as setforth herein. A Table 1A or Table 1B Cas13b effector protein isdiscussed herein in more detail in conjunction with Table 1A or Table1B.

In some embodiment of the non-naturally occurring or engineeredcomposition of the invention, the Cas13b effector protein is associatedwith one or more functional domains. The association can be by directlinkage of the effector protein to the functional domain, or byassociation with the crRNA. In a non-limiting example, the crRNAcomprises an added or inserted sequence that can be associated with afunctional domain of interest, including, for example, an aptamer or anucleotide that binds to a nucleic acid binding adapter protein.

In certain non-limiting embodiments, a non-naturally occurring orengineered composition of the invention comprises a functional domaincleaves the target RNA sequence.

In certain non-limiting embodiments, the non-naturally occurring orengineered composition of the invention comprises a functional domainthat modifies transcription or translation of the target RNA sequence.

In some embodiment of the composition of the invention, the Cas13beffector protein is associated with one or more functional domains; andthe effector protein contains one or more mutations within an HEPNdomain, whereby the complex can deliver an epigenentic modifier or atranscriptional or translational activation or repression signal. Thecomplex can be formed in vitro or ex vivo and introduced into a cell orcontacted with RNA; or can be formed in vivo.

In some embodiment of the non-naturally occurring or engineeredcomposition of the invention, the Cas13b effector protein and theaccessory protein are from the same organism.

In some embodiment of the non-naturally occurring or engineeredcomposition of the invention, the Cas13b effector protein and theaccessory protein are from different organisms.

The invention also provides a Type VI-B CRISPR-Cas vector system, whichcomprises one or more vectors comprising:

-   -   a first regulatory element operably linked to a nucleotide        sequence encoding the Cas13b effector protein, and    -   a second regulatory element operably linked to a nucleotide        sequence encoding the c:rRNA.

In certain embodiments, the vector system of the invention furthercomprises a regulatory element operably linked to a nucleotide sequenceof a Type VI-B CRISPR-Cas accessory protein.

When appropriate, the nucleotide sequence encoding the Type VI-BCRISPR-Cas effector protein and/or the nucleotide sequence encoding theType VI-B CRISPR-Cas accessory protein is codon optimized for expressionin a eukaryotic cell.

In some embodiment of the vector system of the invention, the nucleotidesequences encoding the Cas13b effector protein and the accessory proteinare codon optimized for expression in a eukaryotic cell.

In some embodiment, the vector system of the invention comprises in asingle vector.

In some embodiment of the vector system of the invention, the one ormore vectors comprise viral vectors.

In some embodiment of the vector system of the invention, the one ormore vectors comprise one or more retroviral, lentiviral, adenoviral,adeno-associated or herpes simplex viral vectors.

The invention provides a delivery system configured to deliver a Cas13beffector protein and one or more nucleic acid components of anon-naturally occurring or engineered composition comprising

-   -   i) a Cas13b effector protein, and    -   ii) a crRNA,    -   wherein the crRNA comprises a) a guide sequence that hybridizes        to a target RNA sequence in a cell, and b) a direct repeat        sequence,    -   wherein the Cas13b effector protein forms a complex with the        crRNA,    -   wherein the guide sequence directs sequence-specific binding to        the target RNA sequence,    -   whereby there is formed a CRISPR complex comprising the Cas13b        effector protein complexed with the guide sequence that is        hybridized to the target RNA sequence. The complex can be formed        in vitro or ex vivo and introduced into a cell or contacted with        RNA; or can be formed in vivo.

In some embodiment of the delivery system of the invention, the systemcomprises one or more vectors or one or more polynucleotide molecules,the one or more vectors or polynucleotide molecules comprising one ormore polynucleotide molecules encoding the Cas13b effector protein andone or more nucleic acid components of the non-naturally occurring orengineered composition.

In some embodiment, the delivery system of the invention comprises adelivery vehicle comprising liposome(s), particle(s), exosome(s),microvesicle(s), a gene-gun or one or more viral vector(s).

In some embodiment, the non-naturally occurring or engineeredcomposition of the invention is for use in a therapeutic method oftreatment or in a research program.

In some embodiment, the non-naturally occurring or engineered vectorsystem of the invention is for use in a therapeutic method of treatmentor in a research program.

In some embodiment, the non-naturally occurring or engineered deliverysystem of the invention is for use in a therapeutic method of treatmentor in a research program.

The invention provides a method of modifying expression of a target geneof interest, the method comprising contacting a target RNA with one ormore non-naturally occurring or engineered compositions comprising

-   -   i) a Cas13b effector protein, and    -   ii) a crRNA,    -   wherein the crRNA comprises a) a guide sequence that hybridizes        to a target RNA sequence in a cell, and b) a direct repeat        sequence,    -   wherein the Cas13b effector protein forms a complex with the        crRNA,    -   wherein the guide sequence directs sequence-specific binding to        the target RNA sequence in a cell,    -   whereby there is formed a CRISPR complex comprising the Cas13b        effector protein complexed with the guide sequence that is        hybridized to the target RNA sequence,    -   whereby expression of the target locus of interest is modified.        The complex can be formed in vitro or ex vivo and introduced        into a cell or contacted with RNA; or can be formed in vivo.

In some embodiment, the method of modifying expression of a target geneof interest further comprises contacting the the target RNA with anaccessory protein that enhances Cas13b effector protein activity.

In some embodiment of the method of modifying expression of a targetgene of interest, the accessory protein that enhances Cas13b effectorprotein activity is a csx28 protein.

In some embodiment, the method of modifying expression of a target geneof interest further comprises contacting the the target RNA with anaccessory protein that represses Cas13b effector protein activity.

In some embodiment of the method of modifying expression of a targetgene of interest, the accessory protein that represses Cas13b effectorprotein activity is a csx27 protein.

In some embodiment, the method of modifying expression of a target geneof interest comprises cleaving the target RNA.

In some embodiment, the method of modifying expression of a target geneof interest comprises increasing or decreasing expression of the targetRNA.

In some embodiment of the method of modifying expression of a targetgene of interest, the target gene is in a prokaryotic cell.

In some embodiment of the method of modifying expression of a targetgene of interest, the target gene is in a eukaryotic cell.

The invention provides a cell comprising a modified target of interest,wherein the target of interest has been modified according to any of themethod disclosed herein.

In some embodiment of the invention, the cell is a prokaryotic cell.

In some embodiment of the invention, the cell is a eukaryotic cell.

In some embodiment, modification of the target of interest in a cellresults in:

-   -   a cell comprising altered expression of at least one gene        product;    -   a cell comprising altered expression of at least one gene        product, wherein the expression of the at least one gene product        is increased; or    -   a cell comprising altered expression of at least one gene        product, wherein the expression of the at least one gene product        is decreased.

In some embodiment, the cell is a mammalian cell or a human cell.

The invention provides a cell line of or comprising a cell disclosedherein or a cell modified by any of the methods disclosed herein, orprogeny thereof.

The invention provides a multicellular organism comprising one or morecells disclosed herein or one or more cells modified according to any ofthe methods disclosed herein.

The invention provides a plant or animal model comprising one or morecells disclosed herein or one or more cells modified according to any ofthe methods disclosed herein.

The invention provides a gene product from a cell or the cell line orthe organism or the plant or animal model disclosed herein.

In some embodiment, the amount of gene product expressed is greater thanor less than the amount of gene product from a cell that does not havealtered expression.

The invention provides an isolated Cas13b effector protein, comprisingor consisting essentially of or consisting of or as set forth in Table1A or Table 1B. A Table 1A or Table 1B Cas13b effector protein is asdiscussed in more detail herein in conjunction with Table 1A or Table1B. The invention provides an isolated nucleic acid encoding the Cas13beffector protein. In some embodiments of the invention the isolatednucleic acid comprises DNA sequence and further comprises a sequenceencoding a crRNA. The invention provides an isolated eukaryotic cellcomprising the the nucleic acid encoding the Cas13b effector protein.Thus, herein, “Cas13b effector protein” or “effector protein” or “Cas”or “Cas protein” or “RNA targeting effector protein” or “RNA targetingprotein” or like expressions is to be understood with reference to Table1A or Table 1B and can be read as a Table 1A or Table 1B Cas13b effectorprotein; expressions such as “RNA targeting CRISPR system” are to beunderstood with reference to Table 1A or Table 1B and can be read as aTable 1A or Table 1B Cas13b effector protein CRISPR system; andreferences to guide RNA or sgRNA are to be read in conjunction with theherein-discussion of the Cas13b system crRNA, e.g., that which is sgRNAin other systems may be considered as or akin to crRNA in the instantinvention.

The invention provides a method of identifying the requirements of asuitable guide sequence for the Cas13b effector protein of the invention(e.g., Table 1A or Table 1B), said method comprising:

-   -   (a) selecting a set of essential genes within an organism    -   (b) designing a library of targeting guide sequences capable of        hybridizing to regions the coding regions of these genes as well        as 5′ and 3′ UTRs of these genes    -   (c) generating randomized guide sequences that do not hybridize        to any region within the genome of said organism as control        guides    -   (d) preparing a plasmid comprising the RNA-targeting protein and        a first resistance gene and a guide plasmid library comprising        said library of targeting guides and said control guides and a        second resistance gene,    -   (e) co-introducing said plasmids into a host cell    -   (f) introducing said host cells on a selective medium for said        first and second resistance genes    -   (g) sequencing essential genes of growing host cells    -   (h) determining significance of depletion of cells transformed        with targeting guides by comparing depletion of cells with        control guides; and    -   (i) determining based on the depleted guide sequences the        requirements of a suitable guide sequence.

In one aspect of such method, determining the PFS sequence for suitableguide sequence of the RNA-targeting protein is by comparison ofsequences targeted by guides in depleted cells. In one aspect of suchmethod, the method further comprises comparing the guide abundance forthe different conditions in different replicate experiments. In oneaspect of such method, the control guides are selected in that they aredetermined to show limited deviation in guide depletion in replicateexperiments. In one aspect of such method, the significance of depletionis determined as (a) a depletion which is more than the most depletedcontrol guide; or (b) a depletion which is more than the averagedepletion plus two times the standard deviation for the control guides.In one aspect of such method, the host cell is a bacterial host cell. Inone aspect of such method, the step of co-introducing the plasmids is byelectroporation and the host cell is an electro-competent host cell.

Cas13b

The invention provides a method of modifying sequences associated withor at a target locus of interest, the method comprising delivering tosaid locus a non-naturally occurring or engineered compositioncomprising a Cas13b effector protein and one or more nucleic acidcomponents, wherein the effector protein forms a complex with the one ormore nucleic acid components and upon binding of the said complex to thelocus of interest the effector protein induces the modification of thesequences associated with or at the target locus of interest. In apreferred embodiment, the modification is the introduction of a strandbreak. In a preferred embodiment, the sequences associated with or atthe target locus of interest comprises RNA or consists of RNA.

The invention provides a method of modifying sequences associated withor at a target locus of interest, the method comprising delivering tosaid locus a non-naturally occurring or engineered compositioncomprising a Cas13b effector protein, optionally a small accessoryprotein, and one or more nucleic acid components, wherein the effectorprotein forms a complex with the one or more nucleic acid components andupon binding of the said complex to the locus of interest the effectorprotein induces the modification of the sequences associated with or atthe target locus of interest. In a preferred embodiment, themodification is the introduction of a strand break. In a preferredembodiment, the sequences associated with or at the target locus ofinterest comprises RNA or consists of RNA.

The invention provides a method of modifying sequences associated withor at a target locus of interest, the method comprising delivering tosaid sequences associated with or at the locus a non-naturally occurringor engineered composition comprising a Cas13b loci effector protein andone or more nucleic acid components, wherein the Cas13b effector proteinforms a complex with the one or more nucleic acid components and uponbinding of the said complex to the locus of interest the effectorprotein induces the modification of sequences associated with or at thetarget locus of interest. In a preferred embodiment, the modification isthe introduction of a strand break. In a preferred embodiment the Cas13beffector protein forms a complex with one nucleic acid component;advantageously an engineered or non-naturally occurring nucleic acidcomponent. The induction of modification of sequences associated with orat the target locus of interest can be Cas13b effector protein-nucleicacid guided. In a preferred embodiment the one nucleic acid component isa CRISPR RNA (crRNA). In a preferred embodiment the one nucleic acidcomponent is a mature crRNA or guide RNA, wherein the mature crRNA orguide RNA comprises a spacer sequence (or guide sequence) and a directrepeat (DR) sequence or derivatives thereof. In a preferred embodimentthe spacer sequence or the derivative thereof comprises a seed sequence,wherein the seed sequence is critical for recognition and/orhybridization to the sequence at the target locus. In a preferredembodiment of the invention the crRNA is a short crRNA that may beassociated with a short DR sequence. In another embodiment of theinvention the crRNA is a long crRNA that may be associated with a longDR sequence (or dual DR). Aspects of the invention relate to Cas13beffector protein complexes having one or more non-naturally occurring orengineered or modified or optimized nucleic acid components. In apreferred embodiment the nucleic acid component comprises RNA. In apreferred embodiment the nucleic acid component of the complex maycomprise a guide sequence linked to a direct repeat sequence, whereinthe direct repeat sequence comprises one or more stem loops or optimizedsecondary structures. In preferred embodiments of the invention, thedirect repeat may be a short DR or a long DR (dual DR). In a preferredembodiment the direct repeat may be modified to comprise one or moreprotein-binding RNA aptamers. In a preferred embodiment, one or moreaptamers may be included such as part of optimized secondary structure.Such aptamers may be capable of binding a bacteriophage coat protein.The bacteriophage coat protein may be selected from the group comprisingQβ, F2, GA, fr, JP501, MS2, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1,TW18, VK, SP, FI, ID2, NL95, TW19, AP205, ϕCb5, ϕCb8r, ϕCb12r, ϕCb23r,7s and PRR1. In a preferred embodiment the bacteriophage coat protein isMS2. The invention also provides for the nucleic acid component of thecomplex being 30 or more, 40 or more or 50 or more nucleotides inlength.

The invention provides methods of genome editing or modifying sequencesassociated with or at a target locus of interest wherein the methodcomprises introducing a Cas13b complex into any desired cell type,prokaryotic or eukaryotic cell, whereby the Cas13b effector proteincomplex effectively functions to interfere with RNA in the eukaryotic orprokaryotic cell. In preferred embodiments, the cell is a eukaryoticcell and the RNA is transcribed from a mammalian genome or is present ina mammalian cell. In preferred methods of RNA editing or genome editingin human cells, the Cas13b effector proteins may include but are notlimited to the specific species of Cas13b effector proteins disclosedherein.

The invention also provides a method of modifying a target locus ofinterest, the method comprising delivering to said locus a non-naturallyoccurring or engineered composition comprising a Cas13b effector proteinand one or more nucleic acid components, wherein the Cas13b effectorprotein forms a complex with the one or more nucleic acid components andupon binding of the said complex to the locus of interest the effectorprotein induces the modification of the target locus of interest. In apreferred embodiment, the modification is the introduction of a strandbreak.

In such methods the target locus of interest may be comprised within aRNA molecule. In such methods the target locus of interest may becomprised in a RNA molecule in vitro.

In such methods the target locus of interest may be comprised in a RNAmolecule within a cell. The cell may be a prokaryotic cell or aeukaryotic cell. The cell may be a mammalian cell. The modificationintroduced to the cell by the present invention may be such that thecell and progeny of the cell are altered for improved production ofbiologic products such as an antibody, starch, alcohol or other desiredcellular output. The modification introduced to the cell by the presentinvention may be such that the cell and progeny of the cell include analteration that changes the biologic product produced.

The mammalian cell many be a non-human mammal, e.g., primate, bovine,ovine, porcine, canine, rodent, Leporidae such as monkey, cow, sheep,pig, dog, rabbit, rat or mouse cell. The cell may be a non-mammalianeukaryotic cell such as poultry bird (e.g., chicken), vertebrate fish(e.g., salmon) or shellfish (e.g., oyster, claim, lobster, shrimp) cell.The cell may also be a plant cell. The plant cell may be of a monocot ordicot or of a crop or grain plant such as cassava, corn, sorghum,soybean, wheat, oat or rice. The plant cell may also be of an algae,tree or production plant, fruit or vegetable (e.g., trees such as citrustrees, e.g., orange, grapefruit or lemon trees; peach or nectarinetrees; apple or pear trees; nut trees such as almond or walnut orpistachio trees; nightshade plants; plants of the genus Brassica; plantsof the genus Lactuca; plants of the genus Spinacia; plants of the genusCapsicum; cotton, tobacco, asparagus, carrot, cabbage, broccoli,cauliflower, tomato, eggplant, pepper, lettuce, spinach, strawberry,blueberry, raspberry, blackberry, grape, coffee, cocoa, etc).

The invention provides a method of modifying a target locus of interest,the method comprising delivering to said locus a non-naturally occurringor engineered composition comprising a Cas13b effector protein and oneor more nucleic acid components, wherein the effector protein forms acomplex with the one or more nucleic acid components and upon binding ofthe said complex to the locus of interest the effector protein inducesthe modification of the target locus of interest. In a preferredembodiment, the modification is the introduction of a strand break.

In such methods the target locus of interest may be comprised within anRNA molecule. In a preferred embodiment, the target locus of interestcomprises or consists of RNA.

The invention also provides a method of modifying a target locus ofinterest, the method comprising delivering to said locus a non-naturallyoccurring or engineered composition comprising a Cas13b effector proteinand one or more nucleic acid components, wherein the Cas13b effectorprotein forms a complex with the one or more nucleic acid components andupon binding of the said complex to the locus of interest the effectorprotein induces the modification of the target locus of interest. In apreferred embodiment, the modification is the introduction of a strandbreak.

Preferably, in such methods the target locus of interest may becomprised in a RNA molecule in vitro. Also preferably, in such methodsthe target locus of interest may be comprised in a RNA molecule within acell. The cell may be a prokaryotic cell or a eukaryotic cell. The cellmay be a mammalian cell. The cell may be a rodent cell. The cell may bea mouse cell.

In any of the described methods the target locus of interest may be agenomic or epigenomic locus of interest. In any of the described methodsthe complex may be delivered with multiple guides for multiplexed use.In any of the described methods more than one protein(s) may be used.

In further aspects of the invention the nucleic acid components maycomprise a CRISPR RNA (crRNA) sequence. As the effector protein is aCas13b effector protein, the nucleic acid components may comprise aCRISPR RNA (crRNA) sequence and generally may not comprise anytrans-activating crRNA (tracr RNA) sequence.

In any of the described methods the effector protein and nucleic acidcomponents may be provided via one or more polynucleotide moleculesencoding the protein and/or nucleic acid component(s), and wherein theone or more polynucleotide molecules are operably configured to expressthe protein and/or the nucleic acid component(s). The one or morepolynucleotide molecules may comprise one or more regulatory elementsoperably configured to express the protein and/or the nucleic acidcomponent(s). The one or more polynucleotide molecules may be comprisedwithin one or more vectors. In any of the described methods the targetlocus of interest may be a genomic, epigenomic, or transcriptomic locusof interest. In any of the described methods the complex may bedelivered with multiple guides for multiplexed use. In any of thedescribed methods more than one protein(s) may be used.

In any of the described methods the strand break may be a single strandbreak or a double strand break. In preferred embodiments the doublestrand break may refer to the breakage of two sections of RNA, such asthe two sections of RNA formed when a single strand RNA molecule hasfolded onto itself or putative double helices that are formed with anRNA molecule which contains self-complementary sequences allows parts ofthe RNA to fold and pair with itself.

Regulatory elements may comprise inducible promotors. Polynucleotidesand/or vector systems may comprise inducible systems.

In any of the described methods the one or more polynucleotide moleculesmay be comprised in a delivery system, or the one or more vectors may becomprised in a delivery system.

In any of the described methods the non-naturally occurring orengineered composition may be delivered via liposomes, particlesincluding nanoparticles, exosomes, microvesicles, a gene-gun or one ormore viral vectors.

The invention also provides a non-naturally occurring or engineeredcomposition which is a composition having the characteristics asdiscussed herein or defined in any of the herein described methods.

In certain embodiments, the invention thus provides a non-naturallyoccurring or engineered composition, such as particularly a compositioncapable of or configured to modify a target locus of interest, saidcomposition comprising a Cas13b effector protein and one or more nucleicacid components, wherein the effector protein forms a complex with theone or more nucleic acid components and upon binding of the said complexto the locus of interest the effector protein induces the modificationof the target locus of interest. In certain embodiments, the effectorprotein may be a Cas13b effector protein.

The invention also provides in a further aspect a non-naturallyoccurring or engineered composition, such as particularly a compositioncapable of or configured to modify a target locus of interest, saidcomposition comprising: (a) a guide RNA molecule (or a combination ofguide RNA molecules, e.g., a first guide RNA molecule and a second guideRNA molecule) or a nucleic acid encoding the guide RNA molecule (or oneor more nucleic acids encoding the combination of guide RNA molecules);(b) a Cas13b effector protein. In certain embodiments, the effectorprotein may be a Cas13b effector protein.

The invention also provides in a further aspect a non-naturallyoccurring or engineered composition comprising: (I.) one or moreCRISPR-Cas system polynucleotide sequences comprising (a) a guidesequence capable of hybridizing to a target sequence in a polynucleotidelocus, (b) a tracr mate sequence, and (c) a tracrRNA sequence, and (II.)a second polynucleotide sequence encoding a Cas13b effector protein,wherein when transcribed, the tracr mate sequence hybridizes to thetracrRNA sequence and the guide sequence directs sequence-specificbinding of a CRISPR complex to the target sequence, and wherein theCRISPR complex comprises the Cas13b effector protein complexed with (1)the guide sequence that is hybridized to the target sequence, and (2)the tracr mate sequence that is hybridized to the tracrRNA sequence. Incertain embodiments, the effector protein may be a Cas13b effectorprotein.

In certain embodiments, a tracrRNA may not be required. Hence, theinvention also provides in certain embodiments a non-naturally occurringor engineered composition comprising: (I.) one or more CRISPR-Cas systempolynucleotide sequences comprising (a) a guide sequence capable ofhybridizing to a target sequence in a polynucleotide locus, and (b) adirect repeat sequence, and (II.) a second polynucleotide sequenceencoding a Cas13b effector protein, wherein when transcribed, the guidesequence directs sequence-specific binding of a CRISPR complex to thetarget sequence, and wherein the CRISPR complex comprises the Cas13beffector protein complexed with (1) the guide sequence that ishybridized to the target sequence, and (2) the direct repeat sequence.Preferably, the effector protein may be a Cas13b effector protein.Without limitation, the Applicants hypothesize that in such instances,the direct repeat sequence may comprise secondary structure that issufficient for crRNA loading onto the effector protein. By means ofexample and not limitation, such secondary structure may comprise,consist essentially of or consist of a stem loop (such as one or morestem loops) within the direct repeat.

The invention also provides a vector system comprising one or morevectors, the one or more vectors comprising one or more polynucleotidemolecules encoding components of a non-naturally occurring or engineeredcomposition which is a composition having the characteristics as definedin any of the herein described methods.

The invention also provides a delivery system comprising one or morevectors or one or more polynucleotide molecules, the one or more vectorsor polynucleotide molecules comprising one or more polynucleotidemolecules encoding components of a non-naturally occurring or engineeredcomposition which is a composition having the characteristics discussedherein or as defined in any of the herein described methods.

The invention also provides a non-naturally occurring or engineeredcomposition, or one or more polynucleotides encoding components of saidcomposition, or vector or delivery systems comprising one or morepolynucleotides encoding components of said composition for use in atherapeutic method of treatment. The therapeutic method of treatment maycomprise gene or genome editing, or gene therapy.

The invention also provides for methods and compositions wherein one ormore amino acid residues of the effector protein may be modified e.g.,an engineered or non-naturally-occurring Cas13b effector protein of orcomprising or consisting essentially a Table 1A or Table 1B protein. Inan embodiment, the modification may comprise mutation of one or moreamino acid residues of the effector protein. The one or more mutationsmay be in one or more catalytically active domains of the effectorprotein. The effector protein may have reduced or abolished nucleaseactivity compared with an effector protein lacking said one or moremutations. The effector protein may not direct cleavage of one RNAstrand at the target locus of interest. In a preferred embodiment, theone or more mutations may comprise two mutations. In a preferredembodiment the one or more amino acid residues are modified in theCas13b effector protein, e.g., an engineered or non-naturally-occurringCas13b effector protein. In certain embodiments of the invention theeffector protein comprises one or more HEPN domains. In a preferredembodiment, the effector protein comprises two HEPN domains. In anotherpreferred embodiment, the effector protein comprises one HEPN domain atthe C-terminus and another HEPN domain at the N-terminus of the protein.In certain embodiments, the one or more mutations or the two or moremutations may be in a catalytically active domain of the effectorprotein comprising a HEPN domain, or a catalytically active domain whichis homologous to a HEPN domain. In certain embodiments, the effectorprotein comprises one or more of the following mutations: R116A, H121A,R1177A, H1182A (wherein amino acid positions correspond to amino acidpositions of Group 29 protein originating from Bergeyella zoohelcum ATCC43767). The skilled person will understand that corresponding amino acidpositions in different Cas13b proteins may be mutated to the sameeffect. In certain embodiments, one or more mutations abolish catalyticactivity of the protein completely or partially (e.g. altered cleavagerate, altered specificity, etc.) In certain embodiments, the effectorprotein as described herein is a “dead” effector protein, such as a deadCas13b effector protein (i.e. dCas13b). In certain embodiments, theeffector protein has one or more mutations in HEPN domain 1. In certainembodiments, the effector protein has one or more mutations in HEPNdomain 2. In certain embodiments, the effectyor protein has one or moremutations in HEPN domain 1 and HEPN domain 2. The effector protein maycomprise one or more heterologous functional domains. The one or moreheterologous functional domains may comprise one or more nuclearlocalization signal (NLS) domains. The one or more heterologousfunctional domains may comprise at least two or more NLS domains. Theone or more NLS domain(s) may be positioned at or near or in proximityto a terminus of the effector protein (e.g., Cas13b effector protein)and if two or more NLSs, each of the two may be positioned at or near orin proximity to a terminus of the effector protein (e.g., Cas13beffector protein). The one or more heterologous functional domains maycomprise one or more transcriptional activation domains. In a preferredembodiment the transcriptional activation domain may comprise VP64. Theone or more heterologous functional domains may comprise one or moretranscriptional repression domains. In a preferred embodiment thetranscriptional repression domain comprises a KRAB domain or a SIDdomain (e.g. SID4X). The one or more heterologous functional domains maycomprise one or more nuclease domains. In a preferred embodiment anuclease domain comprises Fok1.

The invention also provides for the one or more heterologous functionaldomains to have one or more of the following activities: methylaseactivity, demethylase activity, transcription activation activity,transcription repression activity, transcription release factoractivity, histone modification activity, nuclease activity,single-strand RNA cleavage activity, double-strand RNA cleavageactivity, single-strand DNA cleavage activity, double-strand DNAcleavage activity and nucleic acid binding activity. At least one ormore heterologous functional domains may be at or near theamino-terminus of the effector protein and/or wherein at least one ormore heterologous functional domains is at or near the carboxy-terminusof the effector protein. The one or more heterologous functional domainsmay be fused to the effector protein. The one or more heterologousfunctional domains may be tethered to the effector protein. The one ormore heterologous functional domains may be linked to the effectorprotein by a linker moiety.

In certain embodiments, the Cas13b effector proteins as intended hereinmay be associated with a locus comprising short CRISPR repeats between30 and 40 bp long, more typically between 34 and 38 bp long, even moretypically between 36 and 37 bp long, e.g., 30, 31, 32, 33, 34, 35, 36,37, 38, 39, or 40 bp long. In certain embodiments the CRISPR repeats arelong or dual repeats between 80 and 350 bp long such as between 80 and200 bp long, even more typically between 86 and 88 bp long, e.g., 80,81, 82, 83, 84, 85, 86, 87, 88, 89, or 90 bp long.

In certain embodiments, a protospacer adjacent motif (PAM) or PAM-likemotif directs binding of the effector protein (e.g. a Cas13b effectorprotein) complex as disclosed herein to the target locus of interest. Insome embodiments, the PAM may be a 5′ PAM (i.e., located upstream of the5′ end of the protospacer). In other embodiments, the PAM may be a 3′PAM (i.e., located downstream of the 5′ end of the protospacer). Inother embodiments, both a 5′ PAM and a 3′ PAM are required. In certainembodiments of the invention, a PAM or PAM-like motif may not berequired for directing binding of the effector protein (e.g. a Cas13beffector protein). In certain embodiments, a 5′ PAM is D (i.e. A, G, orU). In certain embodiments, a 5′ PAM is D for Cas13b effectors. Incertain embodiments of the invention, cleavage at repeat sequences maygenerate crRNAs (e.g. short or long crRNAs) containing a full spacersequence flanked by a short nucleotide (e.g. 5, 6, 7, 8, 9, or 10 nt orlonger if it is a dual repeat) repeat sequence at the 5′ end (this maybe referred to as a crRNA “tag”) and the rest of the repeat at the 3′end. In certain embodiments, targeting by the effector proteinsdescribed herein may require the lack of homology between the crRNA tagand the target 5′ flanking sequence. This requirement may be similar tothat described further in Samai et al. “Co-transcriptional DNA and RNACleavage during Type III CRISPR-Cas Immunity” Cell 161, 1164-1174, May21, 2015, where the requirement is thought to distinguish between bonafide targets on invading nucleic acids from the CRISPR array itself, andwhere the presence of repeat sequences will lead to full homology withthe crRNA tag and prevent autoimmunity.

In certain embodiments, Cas13b effector protein is engineered and cancomprise one or more mutations that reduce or eliminate nucleaseactivity, thereby reducing or eliminating RNA interfering activity.Mutations can also be made at neighboring residues, e.g., at amino acidsnear those that participate in the nuclease activity. In someembodiments, one or more putative catalytic nuclease domains areinactivated and the effector protein complex lacks cleavage activity andfunctions as an RNA binding complex. In a preferred embodiment, theresulting RNA binding complex may be linked with one or more functionaldomains as described herein.

In certain embodiments, the one or more functional domains arecontrollable, i.e. inducible.

In certain embodiments of the invention, the guide RNA or mature crRNAcomprises, consists essentially of, or consists of a direct repeatsequence and a guide sequence or spacer sequence. In certainembodiments, the guide RNA or mature crRNA comprises, consistsessentially of, or consists of a direct repeat sequence linked to aguide sequence or spacer sequence. In preferred embodiments of theinvention, the mature crRNA comprises a stem loop or an optimized stemloop structure or an optimized secondary structure. In preferredembodiments the mature crRNA comprises a stem loop or an optimized stemloop structure in the direct repeat sequence, wherein the stem loop oroptimized stem loop structure is important for cleavage activity. Incertain embodiments, the mature crRNA preferably comprises a single stemloop. In certain embodiments, the direct repeat sequence preferablycomprises a single stem loop. In certain embodiments, the cleavageactivity of the effector protein complex is modified by introducingmutations that affect the stem loop RNA duplex structure. In preferredembodiments, mutations which maintain the RNA duplex of the stem loopmay be introduced, whereby the cleavage activity of the effector proteincomplex is maintained. In other preferred embodiments, mutations whichdisrupt the RNA duplex structure of the stem loop may be introduced,whereby the cleavage activity of the effector protein complex iscompletely abolished.

The CRISPR system as provided herein can make use of a crRNA oranalogous polynucleotide comprising a guide sequence, wherein thepolynucleotide is an RNA, a DNA or a mixture of RNA and DNA, and/orwherein the polynucleotide comprises one or more nucleotide analogs. Thesequence can comprise any structure, including but not limited to astructure of a native crRNA, such as a bulge, a hairpin or a stem loopstructure. In certain embodiments, the polynucleotide comprising theguide sequence forms a duplex with a second polynucleotide sequencewhich can be an RNA or a DNA sequence.

In certain embodiments, the methods make use of chemically modifiedguide RNAs. Examples of guide RNA chemical modifications include,without limitation, incorporation of 2′ O-methyl (M), 2′-O-methyl3′phosphorothioate (MS), or 2′-O-methyl 3′thioPACE (MSP) at one or moreterminal nucleotides. Such chemically modified guide RNAs can compriseincreased stability and increased activity as compared to unmodifiedguide RNAs, though on-target vs. off-target specificity is notpredictable. (See, Hendel, 2015, Nat Biotechnol. 33(9):985-9, doi:10.1038/nbt.3290, published online 29 Jun. 2015). Chemically modifiedguide RNAs further include, without limitation, RNAs withphosphorothioate linkages and locked nucleic acid (LNA) nucleotidescomprising a methylene bridge between the 2′ and 4′ carbons of theribose ring.

The invention also provides for the nucleotide sequence encoding theeffector protein being codon optimized for expression in a eukaryote oreukaryotic cell in any of the herein described methods or compositions.In an embodiment of the invention, the codon optimized effector proteinis any Cas13b effector protein discussed herein and is codon optimizedfor operability in a eukaryotic cell or organism, e.g., such cell ororganism as elsewhere herein mentioned, for instance, withoutlimitation, a yeast cell, or a mammalian cell or organism, including amouse cell, a rat cell, and a human cell or non-human eukaryoteorganism, e.g., plant.

In certain embodiments of the invention, at least one nuclearlocalization signal (NLS) is attached to the nucleic acid sequencesencoding the Cas13b effector proteins. In preferred embodiments at leastone or more C-terminal or N-terminal NLSs are attached (and hencenucleic acid molecule(s) coding for the Cas13b effector protein caninclude coding for NLS(s) so that the expressed product has the NLS(s)attached or connected). In a preferred embodiment a C-terminal NLS isattached for optimal expression and nuclear targeting in eukaryoticcells, preferably human cells. The invention also encompasses methodsfor delivering multiple nucleic acid components, wherein each nucleicacid component is specific for a different target locus of interestthereby modifying multiple target loci of interest. The nucleic acidcomponent of the complex may comprise one or more protein-binding RNAaptamers. The one or more aptamers may be capable of binding abacteriophage coat protein. The bacteriophage coat protein may beselected from the group comprising Qβ, F2, GA, fr, JP501, MS2, M12, R17,BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19,AP205, ϕCb5, ϕCb8r, ϕCb12r, ckCb23r, 7s and PRR1. In a preferredembodiment the bacteriophage coat protein is MS2. The invention alsoprovides for the nucleic acid component of the complex being 30 or more,40 or more or 50 or more nucleotides in length.

In a further aspect, the invention provides a eukaryotic cell comprisinga modified target locus of interest, wherein the target locus ofinterest has been modified according to in any of the herein describedmethods. A further aspect provides a cell line of said cell. Anotheraspect provides a multicellular organism comprising one or more saidcells.

In certain embodiments, the modification of the target locus of interestmay result in: the eukaryotic cell comprising altered expression of atleast one gene product; the eukaryotic cell comprising alteredexpression of at least one gene product, wherein the expression of theat least one gene product is increased; the eukaryotic cell comprisingaltered expression of at least one gene product, wherein the expressionof the at least one gene product is decreased; or the eukaryotic cellcomprising an edited genome.

In certain embodiments, the eukaryotic cell may be a mammalian cell or ahuman cell.

In further embodiments, the non-naturally occurring or engineeredcompositions, the vector systems, or the delivery systems as describedin the present specification may be used for: site-specific geneknockout; site-specific genome editing; RNA sequence-specificinterference; or multiplexed genome engineering.

Also provided is a gene product from the cell, the cell line, or theorganism as described herein. In certain embodiments, the amount of geneproduct expressed may be greater than or less than the amount of geneproduct from a cell that does not have altered expression or editedgenome. In certain embodiments, the gene product may be altered incomparison with the gene product from a cell that does not have alteredexpression or edited genome.

In another aspect, the invention provides a method for identifying novelnucleic acid modifying effectors, comprising: identifying putativenucleic acid modifying loci from a set of nucleic acid sequencesencoding the putative nucleic acid modifying enzyme loci that are withina defined distance from a conserved genomic element of the loci, thatcomprise at least one protein above a defined size limit, or both;grouping the identified putative nucleic acid modifying loci intosubsets comprising homologous proteins; identifying a final set ofcandidate nucleic acid modifying loci by selecting nucleic acidmodifying loci from one or more subsets based on one or more of thefollowing; subsets comprising loci with putative effector proteins withlow domain homology matches to known protein domains relative to loci inother subsets, subsets comprising putative proteins with minimaldistances to the conserved genomic element relative to loci in othersubsets, subsets with loci comprising large effector proteins having asame orientations as putative adjacent accessory proteins relative tolarge effector proteins in other subsets, subset comprising putativeeffector proteins with lower existing nucleic acid modifyingclassifications relative to other loci, subsets comprising loci with alower proximity to known nucleic acid modifying loci relative to othersubsets, and total number of candidate loci in each subset.

In one embodiment, the set of nucleic acid sequences is obtained from agenomic or metagenomic database, such as a genomic or metagenomicdatabase comprising prokaryotic genomic or metagenomic sequences.

In one embodiment, the defined distance from the conserved genomicelement is between 1 kb and 25 kb.

In one embodiment, the conserved genomic element comprises a repetitiveelement, such as a CRISPR array. In a specific embodiment, the defineddistance from the conserved genomic element is within 10 kb of theCRISPR array.

In one embodiment, the defined size limit of a protein comprised withinthe putative nucleic acid modifying (effector) locus is greater than 200amino acids, or more particularly, the defined size limit is greaterthan 700 amino acids. In one embodiment, the putative nucleic acidmodifying locus is between 900 to 1800 amino acids.

In one embodiment, the conserved genomic elements are identified using arepeat or pattern finding analysis of the set of nucleic acids, such asPILER-CR.

In one embodiment, the grouping step of the method described herein isbased, at least in part, on results of a domain homology search or anHHpred protein domain homology search.

In one embodiment, the defined threshold is a BLAST nearest-neighborcut-off value of 0 to 1e-7.

In one embodiment, the method described herein further comprises afiltering step that includes only loci with putative proteins between900 and 1800 amino acids.

In one embodiment, the method described herein further comprisesexperimental validation of the nucleic acid modifying function of thecandidate nucleic acid modifying effectors comprising generating a setof nucleic acid constructs encoding the nucleic acid modifying effectorsand performing one or more biochemical validation assays, such asthrough the use of PAM validation in bacterial colonies, in vitrocleavage assays, the Surveyor method, experiments in mammalian cells,PFS validation, or a combination thereof.

In one embodiment, the method described herein further comprisespreparing a non-naturally occurring or engineered composition comprisingone or more proteins from the identified nucleic acid modifying loci.

In one embodiment, the identified loci comprise a Class 2 CRISPReffector, or the identified loci lack Cas1 or Cas2, or the identifiedloci comprise a single effector.

In one embodiment, the single large effector protein is greater than900, or greater than 1100 amino acids in length, or comprises at leastone HEPN domain.

In one embodiment, the at least one HEPN domain is near a N- orC-terminus of the effector protein, or is located in an interiorposition of the effector protein.

In one embodiment, the single large effector protein comprises a HEPNdomain at the N- and C-terminus and two HEPN domains internal to theprotein.

In one embodiment, the identified loci further comprise one or two smallputative accessory proteins within 2 kb to 10 kb of the CRISPR array.

In one embodiment, a small accessory protein is less than 700 aminoacids. In one embodiment, the small accessory protein is from 50 to 300amino acids in length.

In one embodiment, the small accessory protein comprises multiplepredicted transmembrane domains, or comprises four predictedtransmembrane domains, or comprises at least one HEPN domain.

In one embodiment, the small accessory protein comprises at least oneHEPN domain and at least one transmembrane domain.

In one embodiment, the loci comprise no additional proteins out to 25 kbfrom the CRISPR array.

In one embodiment, the CRISPR array comprises direct repeat sequencescomprising about 36 nucleotides in length. In a specific embodiment, thedirect repeat comprises a GTTG/GUUG at the 5′ end that is reversecomplementary to a CAAC at the 3′ end.

In one embodiment, the CRISPR array comprises spacer sequencescomprising about 30 nucleotides in length.

In one embodiment, the identified loci lack a small accessory protein.

The invention provides a method of identifying novel CRISPR effectors,comprising: a) identifying sequences in a genomic or metagenomicdatabase encoding a CRISPR array; b) identifying one or more OpenReading Frames (ORFs) in said selected sequences within 10 kb of theCRISPR array; c) selecting loci based on the presence of a putativeCRISPR effector protein between 900-1800 amino acids in size, d)selecting loci encoding a putative accessory protein of 50-300 aminoacids; and e) identifying loci encoding a putative CRISPR effector andCRISPR accessory proteins and optionally classifying them based onstructure analysis.

In one embodiment, the CRISPR effector is a Type VI CRISPR effector. Inan embodiment, step (a) comprises i) comparing sequences in a genomicand/or metagenomic database with at least one pre-identified seedsequence that encodes a CRISPR array, and selecting sequences comprisingsaid seed sequence; or ii) identifying CRISPR arrays based on a CRISPRalgorithm.

In an embodiment, step (d) comprises identifying nuclease domains. In anembodiment, step (d) comprises identifying RuvC, HPN, and/or HEPNdomains.

In an embodiment, no ORF encoding Cas1 or Cas2 is present within 10 kbof the CRISPR array.

In an embodiment, an ORF in step (b) encodes a putative accessoryprotein of 50-300 amino acids.

In an embodiment, putative novel CRISPR effectors obtained in step (d)are used as seed sequences for further comparing genomic and/ormetagenomics sequences and subsequent selecting loci of interest asdescribed in steps a) to d) of claim 1. In an embodiment, thepre-identified seed sequence is obtained by a method comprising: (a)identifying CRISPR motifs in a genomic or metagenomic database, (b)extracting multiple features in said identified CRISPR motifs, (c)classifying the CRISPR loci using unsupervised learning, (d) identifyingconserved locus elements based on said classification, and (e) selectingtherefrom a putative CRISPR effector suitable as seed sequence.

In an embodiment, the features include protein elements, repeatstructure, repeat sequence, spacer sequence and spacer mapping. In anembodiment, the genomic and metagenomic databases are bacterial and/orarchaeal genomes. In an embodiment, the genomic and metagenomicsequences are obtained from the Ensembl and/or NCBI genome databases. Inan embodiment, the structure analysis in step (d) is based on secondarystructure prediction and/or sequence alignments. In an embodiment, step(d) is achieved by clustering of the remaining loci based on theproteins they encode and manual curation of the obtained clusters.

Accordingly, it is an object of the invention not to encompass withinthe invention any previously known product, process of making theproduct, or method of using the product such that Applicants reserve theright and hereby disclose a disclaimer of any previously known product,process, or method. It is further noted that the invention does notintend to encompass within the scope of the invention any product,process, or making of the product or method of using the product, whichdoes not meet the written description and enablement requirements of theUSPTO (35 U.S.C. § 112, first paragraph) or the EPO (Article 83 of theEPC), such that Applicants reserve the right and hereby disclose adisclaimer of any previously described product, process of making theproduct, or method of using the product. It may be advantageous in thepractice of the invention to be in compliance with Art. 53(c) EPC andRule 28(b) and (c) EPC. Nothing herein is to be construed as a promise.

It is noted that in this disclosure and particularly in the claimsand/or paragraphs, terms such as “comprises”, “comprised”, “comprising”and the like can have the meaning attributed to it in U.S. Patent law;e.g., they can mean “includes”, “included”, “including”, and the like;and that terms such as “consisting essentially of” and “consistsessentially of” have the meaning ascribed to them in U.S. Patent law,e.g., they allow for elements not explicitly recited, but excludeelements that are found in the prior art or that affect a basic or novelcharacteristic of the invention.

These and other embodiments are disclosed or are obvious from andencompassed by, the following Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1A-1B shows a tree alignment of Cas13b orthologs.

FIG. 2A-2C shows a tree alignment of C2c2 and Cas13b orthologs.

FIG. 3 shows an exemplary result of the testing of Cas13b orthologs foractivity in E. Coli, whereby the introduction of Cas13b from B.zoohelcum is compared to the introduction of an empty vector.

FIG. 4 shows a general comparison of the specific RNA cleavage activityobtained with the different orthologs of Table 1A or Table 1B.

FIG. 5 shows an alignment of different Cas13b orthologs as provided inFIGS. 1 and 2 .

FIG. 6A-6B (A) shows the protospacer design for MS2 phage drop plaqueassay to test RNA interference and identify PFS. (B) shows RNAinterference assay schematic. A target sequence is placed in-frame atthe start of the transcribed bla gene that confers ampicillin resistanceor in a non-transcribed region on the opposite strand of the same targetplasmid. Target plasmids were co-transformed with bzcas13b plasmid orempty vectors conferring chloramphenicol resistance and plated on doubleselection antibiotic plates. Depleted colonies were identified andcorresponding targets sequenced for PFS identification.

FIG. 7 shows the heatmap of the normalized PFS score from safelydepleted spacers for orthologs 1, 13 and 16 in the absence and presenceof the csx27 accessory protein.

FIG. 8 shows the heatmap of the normalized PFS score from safelydepleted spacers for orthologs 2, 3, 8, 9, 14, 19 and 21 in the absenceand presence of the csx28 accessory protein.

FIG. 9 shows the heatmap of the normalized PFS score from safelydepleted spacers for orthologs 5, 6, 7, 10, 12 and 15.

FIG. 10A-10BB shows the heatmap of the normalized PFS score from safelydepleted spacers for different orthologs and the derived PFS.

FIG. 11 provides an overview of the luciferase interference data for theCas13b orthologs that were less active in mammalian cells with thetested guides.

FIG. 12 provides an overview of the luciferase interference data for theCas13b orthologs that showed low to intermediate activity in mammaliancells with the tested guides.

FIG. 13 provides an overview of the luciferase interference data forsome of the Cas13b orthologs that showed significant activity inmammalian cells with the tested guides.

FIG. 14A-14B provides an overview of the luciferase interference datafor some of the Cas13b orthologs that showed significant activity inmammalian cells with the tested guides.

FIG. 15 provides an overview of the luciferase interference data for theCas13b orthologs that showed significant activity in mammalian cellswith the tested guides and comparison with C2c2 activity.

FIG. 16 shows the composite data from orthologs having significantactivity in eukaryotic cells with the same guide sequences.

FIG. 17A-17G shows the collateral effect of the Cas13b orthologs inmammalian cells using two different reporter genes, G-luciferase andC-luciferase.

FIG. 18 shows characterization of a highly active Cas13b ortholog forRNA knockdown. A) Schematic of stereotypical Cas13 loci andcorresponding crRNA structure. B) Evaluation of 19 Cas13a, 15 Cas13b,and 7 Cas13c orthologs for luciferase knockdown using two differentguides. Orthologs with efficient knockdown using both guides are labeledwith their host organism name. C) PspCas13b and LwaCas13a knockdownactivity are compared by tiling guides against Gluc and measuringluciferase expression. D) PspCas13b and LwaCas13a knockdown activity arecompared by tiling guides against Cluc and measuring luciferaseexpression. E) Expression levels in log 2(transcripts per million (TPM))values of all genes detected in RNA-seq libraries of non-targetingcontrol (x-axis) compared to Gluc-targeting condition (y-axis) forLwaCas13a (red) and shRNA (black). Shown is the mean of three biologicalreplicates. The Gluc transcript data point is labeled. F) Expressionlevels in log 2(transcripts per million (TPM)) values of all genesdetected in RNA-seq libraries of non-targeting control (x-axis) comparedto Gluc-targeting condition (y-axis) for PspCas13b (blue) and shRNA(black). Shown is the mean of three biological replicates. The Gluctranscript data point is labeled. G) Number of significant off-targetsfrom Gluc knockdown for LwaCas13a, PspCas13b, and shRNA from thetranscriptome wide analysis in E and F.

FIG. 19 shows engineering dCas13b-ADAR fusions for RNA editing. A)Schematic of RNA editing by dCas13b-ADAR fusion proteins. B) Schematicof Cypridina luciferase W85X target and targeting guide design. C)Quantification of luciferase activity restoration for Cas13b-dADAR1(left) and Cas13b-ADAR2-cd (right) with tiling guides of length 30, 50,70, or 84 nt. D) Schematic of target site for targeting Cypridinaluciferase W85X. E) Sequencing quantification of A≥I editing for 50 ntguides targeting Cypridina luciferase W85X.

FIG. 20 shows measuring sequence flexibility for RNA editing byREPAIRv1. A) Schematic of screen for determining Protospacer FlankingSite (PFS) preferences of RNA editing by REPAIRv1. B) Distributions ofRNA editing efficiencies for all 4-N PFS combinations at two differentediting sites C) Quantification of the percent editing of REPAIRv1 atCluc W85 across all possible 3 base motifs. D) Heatmap of 5′ and 3′ basepreferences of RNA editing at Cluc W85 for all possible 3 base motifs.

FIG. 21 shows correction of disease-relevant mutations with REPAIRv1. A)Schematic of target and guide design for targeting AVPR2 878G>A. B) The878G>A mutation in AVPR2 is corrected to varying percentages usingREPAIRv1 with three different guide designs. C) Schematic of target andguide design for targeting FANCC 1517G>A. D) The 1517G>A mutation inFANCC is corrected to varying percentages using REPAIRv1 with threedifferent guide designs. E) Quantification of the percent editing of 34different disease-relevant G>A mutations using REPAIRv1. F) Analysis ofall the possible G>A mutations that could be corrected as annotated bythe ClinVar database. G) The distribution of editing motifs for all G>Amutations in ClinVar is shown versus the editing efficiency by REPAIRv1per motif as quantified on the Gluc transcript.

FIG. 22 shows characterizing specificity of REPAIRv1. A) Schematic ofKRAS target site and guide design. B) Quantification of percent editingfor tiled KRAS-targeting guides. Editing percentages are shown at theon-target and neighboring adenosine sites. For each guide, the region ofduplex RNA is indicated by a red rectangle. C) Transcriptome-wide sitesof significant RNA editing by REPAIRv1 with Cluc targeting guide. Theon-target site Cluc site (254 A>G) is highlighted in orange. D)Transcriptome-wide sites of significant RNA editing by REPAIRv1 withnon-targeting guide.

FIG. 23 shows rational mutagenesis of ADAR2 to improve the specificityof REPAIRv1. A) Quantification of luciferase signal restoration byvarious dCas13-ADAR2 mutants as well as their specificity score plottedalong a schematic for the contacts between key ADAR2 deaminase residuesand the dsRNA target. The specificity score is defined as the ratio ofthe luciferase signal between targeting guide and non-targeting guideconditions. B) Quantification of luciferase signal restoration byvarious dCas13-ADAR2 mutants versus their specificity score. C)Measurement of the on-target editing fraction as well as the number ofsignificant off-targets for each dCas13-ADAR2 mutant by transcriptomewide sequencing of mRNAs. D) Transcriptome-wide sites of significant RNAediting by REPAIRv1 and REPAIRv2 with a guide targeting a preterminationsite in Cluc. The on-target Cluc site (254 A>G) is highlighted inorange. E) RNA sequencing reads surrounding the on-target Cluc editingsite (254 A>G) highlighting the differences in off-target editingbetween REPAIRv1 and REPAIRv2. All A>G edits are highlighted in redwhile sequencing errors are highlighted in blue. F) RNA editing byREPAIRv1 and REPAIRv2 with guides targeting an out-of-frame UAG site inthe endogenous KRAS and PPIB transcripts. The on-target editing fractionis shown as a sideways bar chart on the right for each condition row.The duplex region formed by the guide RNA is shown by a red outline box.

FIG. 24 shows bacterial screening of Cas13b orthologs for in vivoefficiency and PFS determination. A) Schematic of bacterial assay fordetermining the PFS of Cas13b orthologs. Cas13b orthologs withbeta-lactamase targeting spacers are co-transformed with beta-lactamaseexpression plasmids and subjected to double selection. B) Quantitationof interference activity of Cas13b orthologs targeting beta-lactamase asmeasured by colony forming units (cfu). C) PFS logos for Cas13borthologs as determined by depleted sequences from the bacterial assay.

FIG. 25 shows optimization of Cas13b knockdown and furthercharacterization of mismatch specificity. A) Gluc knockdown with twodifferent guides is measured using the top 2 Cas13a and top 4 Cas13borthologs fused to a variety of nuclear localization and nuclear exporttags. B) Knockdown of KRAS is measured for LwaCas13a, RanCas13b,PguCas13b, and PspCas13b with four different guides and compared to fourposition-matched shRNA controls. C) Schematic of the single and doublemismatch plasmid libraries used for evaluating the specificity ofLwaCas13a and PspCas13b knockdown. Every possible single and doublemismatch is present in the target sequence as well as in 3 positionsdirectly flanking the 5′ and 3′ ends of the target site. D) Thedepletion level of transcripts with the indicated single mismatches areplotted as a heatmap for both the LwaCas13a and PspCas13b conditions. E)The depletion level of transcripts with the indicated double mismatchesare plotted as a heatmap for both the LwaCas13a and PspCas13bconditions.

FIG. 26 shows characterization of design parameters for dCas13-ADAR2 RNAediting. A) Knockdown efficiency of Gluc targeting for wildtype Cas13band catalytically inactive H133A/H1058A Cas13b (dCas13b). B)Quantification of luciferase activity restoration by dCas13b fused toeither the wildtype ADAR2 catalytic domain or the hyperactive E488Qmutant ADAR2 catalytic catalytic domain, tested with tiling Cluctargeting guides. C) Guide design and sequencing quantification of A≥Iediting for 30 nt guides targeting Cypridinia luciferase W85X. D) Guidedesign and sequencing quantification of A≥I editing for 50 nt guidestargeting PPIB. E) Influence of linker choice on luciferase activityrestoration by REPAIRv1. F) Influence of base identify opposite thetargeted adenosine on luciferase activity restoration by REPAIRv1.

FIG. 27 shows clinVar motif distribution for G>A mutations. The numberof each possible triplet motif observed in the ClinVar database for allG>A mutations.

FIG. 28A-28B shows RNA binding by truncations of dCas13b. VariousN-terminal and C-terminal truncations of dCas13b are depicted. RNAbinding is indicated where there is ADAR-dependent RNA editing asmeasured by restoration of luciferase signal, comparing activity usingtargeting and non-targeting guides. Amino acid positions correspond toamino acid positions of Prevotella sp. P5-125 Cas13b protein.

FIG. 29 shows comparison of other programmable ADAR systems with thedCas13-ADAR2 editor. A) Schematic of two programmable ADAR schemes:BoxB-based targeting and full length ADAR2 targeting. In the BoxB scheme(top), the ADAR2 deaminase domain (ADAR2_(DD)(E488Q)) is fused to asmall bacterial virus protein called lambda N (

N), which binds specifically a small RNA sequence called BoxB-

. A guide RNA containing two BoxB-

, hairpins can then guide the ADAR2_(DD)(E488Q), -

for site specific editing. In the full length ADAR2 scheme (bottom), thedsRNA binding domains of ADAR2 bind a hairpin in the guide RNA, allowingfor programmable ADAR2 editing. B) Transcriptome-wide sites ofsignificant RNA editing by BoxB-ADAR2_(DD)(E488Q) with a guide targetingCluc and a non-targeting guide. The on-target Cluc site (254 A>G) ishighlighted in orange. C) Transcriptome-wide sites of significant RNAediting by ADAR2 with a guide targeting Cluc and a non-targeting guide.The on-target Cluc site (254 A>G) is highlighted in orange. D)Transcriptome-wide sites of significant RNA editing by REPAIRv1 with aguide targeting Cluc and a non-targeting guide. The on-target Cluc site(254 A>G) is highlighted in orange. E) Quantitation of on-target editingrate percentage for BoxB-ADAR2_(DD)(E488Q), ADAR2, and REPAIRv1 fortargeting guides against Cluc.F) Overlap of off-target sites betweendifferent targeting and non-targeting conditions for programmable ADARsystems.

FIG. 30 shows efficiency and specificity of dCas13b-ADAR2 mutants. A)Quantitation of luciferase activity restoration bydCas13b-ADAR2_(DD)(E488Q) mutants for Cluc-targeting and non-targetingguides. B) Relationship between the ratio of targeting and non-targetingguides and the number of RNA-editing off-targets as quantified bytranscriptome-wide sequencing. C) Quantification of number oftranscriptome-wide off-target RNA editing sites versus on-target Clucediting efficiency for dCas13b-ADAR2_(DD)(E488Q) mutants.

FIG. 31 shows transcriptome-wide specificity of RNA editing bydCas13b-ADAR2_(DD)(E488Q) mutants. A) Transcriptome-wide sites ofsignificant RNA editing by dCas13b-ADAR2_(DD)(E488Q) mutants with aguide targeting Cluc. The on-target Cluc site (254 A>G) is highlightedin orange. B) Transcriptome-wide sites of significant RNA editing bydCas13b-ADAR2_(DD)(E488Q) mutants with a non-targeting guide.

FIG. 32 shows characterization of motif biases in the off-targets ofdCas13b-ADAR2_(DD)(E488Q) editing. A) For each dCas13b-ADAR2_(DD)(E488Q)mutant, the motif present across all A>G off-target edits in thetranscriptome is shown. B) The distribution of off-target A>G edits permotif identity is shown for REPAIRv1 with targeting and non-targetingguide. C) The distribution of off-target A>G edits per motif identity isshown for REPAIRv2 with targeting and non-targeting guide.

FIG. 33 shows further characterization of REPAIRv1 and REPAIRv2off-targets. A) Histogram of the number of off-targets per transcriptfor REPAIRv1. B) Histogram of the number of off-targets per transcriptfor REPAIRv2. C) Variant effect prediction of REPAIRv1 off targets. D)Distribution of potential oncogenic effects of REPAIRv1 off targets. E)Variant effect prediction of REPAIRv2 off targets. F) Distribution ofpotential oncogenic effects of REPAIRv2 off targets.

FIG. 34 shows RNA editing efficiency and specificity of REPAIRv1 andREPAIRv2. A) Quantification of percent editing of KRAS withKRAS-targeting guide 1 at the targeted adenosine and neighboring sitesfor REPAIRv1 and REPAIRv2. B) Quantification of percent editing of KRASwith KRAS-targeting guide 3 at the targeted adenosine and neighboringsites for REPAIRv1 and REPAIRv2. C) Quantification of percent editing ofPPIB with PPIB-targeting guide 2 at the targeted adenosine andneighboring sites for REPAIRv1 and REPAIRv2.

FIG. 35 shows demonstration of all potential codon changes with a A>GRNA editor. A) Table of all potential codon transitions enabled by A>Iediting. B) A codon table demonstrating all the potential codontransitions enabled by A>I editing. C)

FIG. 36A-36B shows effect of Csx proteins on RNA interference. A)Comparison of Cas13b and Cas13b+Csx27 interference; B) Comparison ofCas13b and Cas13b+Csx28 interference.

FIG. 37A-37F show comparison transcript knockdown by Cas13b andCas13b+Csx28 via luciferase assay. A) Pin Cas13b (WP_036860899); B) PbuCas13b (WP_004343973); C) Rin Cas13b (WP_004919755); D) Pau Cas13b(WP_025000926); E) Pgu Cas13b (WP_039434803); F) Pig Cas13b(WP_053444417).

The figures herein are for illustrative purposes only and are notnecessarily drawn to scale.

DETAILED DESCRIPTION OF THE INVENTION

In general, the CRISPR-Cas or CRISPR system refers collectively totranscripts and other elements involved in the expression of ordirecting the activity of CRISPR-associated (“Cas”) genes, includingsequences encoding a Cas gene, a tracr (trans-activating CRISPR)sequence (e.g. tracrRNA or an active partial tracrRNA), a tracr-matesequence (encompassing a “direct repeat” and a tracrRNA-processedpartial direct repeat in the context of an endogenous CRISPR system), aguide sequence (also referred to as a “spacer” in the context of anendogenous CRISPR system), or “RNA(s)” as that term is herein used(e.g., RNA(s) to guide Cas, such as Cas9, e.g. CRISPR RNA andtransactivating (tracr) RNA or a single guide RNA (sgRNA) (chimericRNA)) or other sequences and transcripts from a CRISPR locus. Ingeneral, a CRISPR system is characterized by elements that promote theformation of a CRISPR complex at the site of a target sequence (alsoreferred to as a protospacer in the context of an endogenous CRISPRsystem). When the CRISPR protein is a Class 2 Type VI-B effector, atracrRNA is not required. In an engineered system of the invention, thedirect repeat may encompass naturally-occurring sequences ornonnaturally-occurring sequences. The direct repeat of the invention isnot limited to naturally occurring lengths and sequences. A directrepeat can be 36 nt in length, but a longer or shorter direct repeat canvary. For example, a direct repeat can be 30 nt or longer, such as30-100 nt or longer. For example, a direct repeat can be 30 nt, 40 nt,50 nt, 60 nt, 70 nt, 70 nt, 80 nt, 90 nt, 100 nt or longer in length. Insome embodiments, a direct repeat of the invention can include syntheticnucleotide sequences inserted between the 5′ and 3′ ends of naturallyoccurring direct repeats. In certain embodiments, the inserted sequencemay be self-complementary, for example, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, or 100% self complementary. Furthermore, a direct repeat ofthe invention may include insertions of nucleotides such as an aptameror sequences that bind to an adapter protein (for association withfunctional domains). In certain embodiments, one end of a direct repeatcontaining such an insertion is roughly the first half of a short DR andthe end is roughly the second half of the short DR.

In the context of formation of a CRISPR complex, “target sequence”refers to a sequence to which a guide sequence is designed to havecomplementarity, where hybridization between a target sequence and aguide sequence promotes the formation of a CRISPR complex. A targetsequence may comprise any polynucleotide, such as DNA or RNApolynucleotides. In some embodiments, a target sequence is located inthe nucleus or cytoplasm of a cell. In some embodiments, direct repeatsmay be identified in silico by searching for repetitive motifs thatfulfill any or all of the following criteria: 1. found in a 2 Kb windowof genomic sequence flanking the type II CRISPR locus; 2. span from 20to 50 bp; and 3. interspaced by 20 to 50 bp. In some embodiments, 2 ofthese criteria may be used, for instance 1 and 2, 2 and 3, or 1 and 3.In some embodiments, all 3 criteria may be used.

In embodiments of the invention the terms guide sequence and guide RNA,i.e. RNA capable of guiding Cas13b effector proteins to a target locus,are used interchangeably as in herein cited documents such as WO2014/093622 (PCT/US2013/074667). In general, a guide sequence (or spacersequence) is any polynucleotide sequence having sufficientcomplementarity with a target polynucleotide sequence to hybridize withthe target sequence and direct sequence-specific binding of a CRISPRcomplex to the target sequence. In some embodiments, the degree ofcomplementarity between a guide sequence and its corresponding targetsequence, when optimally aligned using a suitable alignment algorithm,is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%,99%, or more. Optimal alignment may be determined with the use of anysuitable algorithm for aligning sequences, non-limiting example of whichinclude the Smith-Waterman algorithm, the Needleman-Wunsch algorithm,algorithms based on the Burrows-Wheeler Transform (e.g. the BurrowsWheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (NovocraftTechnologies; available at www.novocraft.com), ELAND (Illumina, SanDiego, CA), SOAP (available at soap.genomics.org.cn), and Maq (availableat maq.sourceforge.net). In some embodiments, a guide sequence (orspacer sequence) is about or more than about 5, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45,50, 75, or more nucleotides in length. In some embodiments, a guidesequence is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, orfewer nucleotides in length. Preferably the guide sequence is 10-40nucleotides long, such as 20-30 or 20-40 nucleotides long or longer,such as 30 nucleotides long or about 30 nucleotides long. In certainembodiments, the guide sequence is 10-30 nucleotides long, such as 20-30or 20-40 nucleotides long or longer, such as 30 nucleotides long orabout 30 nucleotides long for Cas13b effectors. In certain embodiments,the guide sequence is 10-30 nucleotides long, such as 20-30 nucleotideslong, such as 30 nucleotides long. The ability of a guide sequence todirect sequence-specific binding of a CRISPR complex to a targetsequence may be assessed by any suitable assay. For example, thecomponents of a CRISPR system sufficient to form a CRISPR complex,including the guide sequence to be tested, may be provided to a hostcell having the corresponding target sequence, such as by transfectionwith vectors encoding the components of the CRISPR sequence, followed byan assessment of preferential cleavage within the target sequence, suchas by Surveyor assay as described herein. Similarly, cleavage of atarget polynucleotide sequence may be evaluated in a test tube byproviding the target sequence, components of a CRISPR complex, includingthe guide sequence to be tested and a control guide sequence differentfrom the test guide sequence, and comparing binding or rate of cleavageat the target sequence between the test and control guide sequencereactions. Other assays are possible, and will occur to those skilled inthe art.

The instant invention provides particular Cas13b effectors, nucleicacids, systems, vectors, and methods of use. All VI-B aredistinguishable from VI-A (Cas13b) by structure, and also by thelocation of the HEPN domains). There appears little that may separateCas13b lacking the small accessory protein from Cas13b which has it,except that VI-B2 loci appear to have the small accessory protein muchmore conserved.

As used herein, the terms Cas13b-s1 accessory protein, Cas13b-s1protein, Cas13b-s1, Csx27, and Csx27 protein are used interchangeablyand the terms Cas13b-s2 accessory protein, Cas13b-s2 protein, Cas13b-S2,Csx28, and Csx28 protein are used interchangeably.

In a classic CRISPR-Cas systems, the degree of complementarity between aguide sequence and its corresponding target sequence can be about ormore than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or 100%;a guide or RNA or crRNA can be about or more than about 5, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,35, 40, 45, 50, 75, or more nucleotides in length; or guide or RNA orcrRNA can be less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, orfewer nucleotides in length; and advantageously tracr RNA is 30 or 50nucleotides in length. However, an aspect of the invention is to reduceoff-target interactions, e.g., reduce the guide interacting with atarget sequence having low complementarity. Indeed, in the examples, itis shown that the invention involves mutations that result in theCRISPR-Cas system being able to distinguish between target andoff-target sequences that have greater than 80% to about 95%complementarity, e.g., 83%-84% or 88-89% or 94-95% complementarity (forinstance, distinguishing between a target having 18 nucleotides from anoff-target of 18 nucleotides having 1, 2 or 3 mismatches). Accordingly,in the context of the present invention the degree of complementaritybetween a guide sequence and its corresponding target sequence isgreater than 94.5% or 95% or 95.5% or 96% or 96.5% or 97% or 97.5% or98% or 98.5% or 99% or 99.5% or 99.9%, or 100%. Off target is less than100% or 99.9% or 99.5% or 99% or 99% or 98.5% or 98% or 97.5% or 97% or96.5% or 96% or 95.5% or 95% or 94.5% or 94% or 93% or 92% or 91% or 90%or 89% or 88% or 87% or 86% or 85% or 84% or 83% or 82% or 81% or 80%complementarity between the sequence and the guide, with it advantageousthat off target is 100% or 99.9% or 99.5% or 99% or 99% or 98.5% or 98%or 97.5% or 97% or 96.5% or 96% or 95.5% or 95% or 94.5% complementaritybetween the sequence and the guide.

In particularly preferred embodiments according to the invention, theguide RNA (capable of guiding Cas to a target locus) may comprise (1) aguide sequence capable of hybridizing to a target locus (apolynucleotide target locus, such as an RNA target locus) in theeukaryotic cell; (2) a direct repeat (DR) sequence) which reside in asingle RNA, i.e. an sgRNA (arranged in a 5′ to 3′ orientation) or crRNA.

In particular embodiments, the wildtype Cas13b effector protein has RNAbinding and cleaving function.

In particular embodiments, the Cas13b effector protein may have DNAcleaving function. In these embodiments, methods may be provided basedon the effector proteins provided herein which comprehend inducing oneor more mutations in a eukaryotic cell (in vitro, i.e. in an isolatedeukaryotic cell) as herein discussed comprising delivering to cell avector as herein discussed. The mutation(s) can include theintroduction, deletion, or substitution of one or more nucleotides ateach target sequence of cell(s) via the guide(s) RNA(s) or sgRNA(s) orcrRNA(s). The mutations can include the introduction, deletion, orsubstitution of 1-75 nucleotides at each target sequence of said cell(s)via the guide(s) RNA(s) or sgRNA(s) or crRNA(s). The mutations caninclude the introduction, deletion, or substitution of 1, 5, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,35, 40, 45, 50, or 75 nucleotides at each target sequence of saidcell(s) via the guide(s) RNA(s) or sgRNA(s) or crRNA(s). The mutationscan include the introduction, deletion, or substitution of 5, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 35, 40, 45, 50, or 75 nucleotides at each target sequence of saidcell(s) via the guide(s) RNA(s) or sgRNA(s) or crRNA(s). The mutationsinclude the introduction, deletion, or substitution of 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35,40, 45, 50, or 75 nucleotides at each target sequence of said cell(s)via the guide(s) RNA(s) or sgRNA(s) or crRNA(s). The mutations caninclude the introduction, deletion, or substitution of 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or 75 nucleotides at eachtarget sequence of said cell(s) via the guide(s) RNA(s) or sgRNA(s) orcrRNA(s). The mutations can include the introduction, deletion, orsubstitution of 40, 45, 50, 75, 100, 200, 300, 400 or 500 nucleotides ateach target sequence of said cell(s) via the guide(s) RNA(s) or sgRNA(s)or crRNAs.

For minimization of toxicity and off-target effect, it will be importantto control the concentration of Cas13b mRNA and guide RNA delivered.Optimal concentrations of Cas13b mRNA and guide RNA can be determined bytesting different concentrations in a cellular or non human eukaryoteanimal model and using deep sequencing the analyze the extent ofmodification at potential off-target genomic loci. Alternatively, tominimize the level of toxicity and off-target effect, Cas13b nickasemRNA (for example S. pyogenes Cas9 with the D10A mutation) can bedelivered with a pair of guide RNAs targeting a site of interest. Guidesequences and strategies to minimize toxicity and off-target effects canbe as in WO 2014/093622 (PCT/US2013/074667); or, via mutation as herein.

Typically, in the context of an endogenous CRISPR system, formation of aCRISPR complex (comprising a guide sequence hybridized to a targetsequence and complexed with one or more Cas proteins) results incleavage of one or both strands (if applicable) in or near (e.g. within1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from) thetarget sequence.

The nucleic acid molecule encoding a Cas13b is advantageously codonoptimized. An example of a codon optimized sequence, is in this instancea sequence optimized for expression in a eukaryote, e.g., humans (i.e.being optimized for expression in humans), or for another eukaryote,animal or mammal as herein discussed; see, e.g., SaCas9 human codonoptimized sequence in WO 2014/093622 (PCT/US2013/074667). Whilst this ispreferred, it will be appreciated that other examples are possible andcodon optimization for a host species other than human, or for codonoptimization for specific organs is known. In some embodiments, anenzyme coding sequence encoding a Cas is codon optimized for expressionin particular cells, such as eukaryotic cells. The eukaryotic cells maybe those of or derived from a particular organism, such as a mammal,including but not limited to human, or non-human eukaryote or animal ormammal as herein discussed, e.g., mouse, rat, rabbit, dog, livestock, ornon-human mammal or primate. In some embodiments, processes formodifying the germ line genetic identity of human beings and/orprocesses for modifying the genetic identity of animals which are likelyto cause them suffering without any substantial medical benefit to manor animal, and also animals resulting from such processes, may beexcluded. In general, codon optimization refers to a process ofmodifying a nucleic acid sequence for enhanced expression in the hostcells of interest by replacing at least one codon (e.g. about or morethan about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of thenative sequence with codons that are more frequently or most frequentlyused in the genes of that host cell while maintaining the native aminoacid sequence. Various species exhibit particular bias for certaincodons of a particular amino acid. Codon bias (differences in codonusage between organisms) often correlates with the efficiency oftranslation of messenger RNA (mRNA), which is in turn believed to bedependent on, among other things, the properties of the codons beingtranslated and the availability of particular transfer RNA (tRNA)molecules. The predominance of selected tRNAs in a cell is generally areflection of the codons used most frequently in peptide synthesis.Accordingly, genes can be tailored for optimal gene expression in agiven organism based on codon optimization. Codon usage tables arereadily available, for example, at the “Codon Usage Database” availableat www.kazusa.or.jp/codon/and these tables can be adapted in a number ofways. See Nakamura, Y., et al. “Codon usage tabulated from theinternational DNA sequence databases: status for the year 2000” Nucl.Acids Res. 28:292 (2000). Computer algorithms for codon optimizing aparticular sequence for expression in a particular host cell are alsoavailable, such as Gene Forge (Aptagen; Jacobus, PA), are alsoavailable. In some embodiments, one or more codons (e.g. 1, 2, 3, 4, 5,10, 15, 20, 25, 50, or more, or all codons) in a sequence encoding a Cascorrespond to the most frequently used codon for a particular aminoacid.

In certain embodiments, the methods as described herein may compriseproviding a Cas13b transgenic cell in which one or more nucleic acidsencoding one or more guide RNAs are provided or introduced operablyconnected in the cell with a regulatory element comprising a promoter ofone or more gene of interest. As used herein, the term “Cas13btransgenic cell” refers to a cell, such as a eukaryotic cell, in which aCas13b gene has been genomically integrated. The nature, type, or originof the cell are not particularly limiting according to the presentinvention. Also the way how the Cas13b transgene is introduced in thecell is may vary and can be any method as is known in the art. Incertain embodiments, the Cas13b transgenic cell is obtained byintroducing the Cas13b transgene in an isolated cell. In certain otherembodiments, the Cas13b transgenic cell is obtained by isolating cellsfrom a Cas13b transgenic organism. By means of example, and withoutlimitation, the Cas13b transgenic cell as referred to herein may bederived from a Cas13b transgenic eukaryote, such as a Cas13b knock-ineukaryote. Reference is made to WO 2014/093622 (PCT/US13/74667),incorporated herein by reference. Methods of US Patent Publication Nos.20120017290 and 20110265198 assigned to Sangamo BioSciences, Inc.directed to targeting the Rosa locus may be modified to utilize theCRISPR Cas system of the present invention. Methods of US PatentPublication No. 20130236946 assigned to Cellectis directed to targetingthe Rosa locus may also be modified to utilize the CRISPR Cas system ofthe present invention. By means of further example reference is made toPlatt et. al. (Cell; 159(2):440-455 (2014)), describing a Cas9 knock-inmouse, which is incorporated herein by reference. The Cas13b transgenecan further comprise a Lox-Stop-polyA-Lox(LSL) cassette therebyrendering Cas13b expression inducible by Cre recombinase. Alternatively,the Cas13b transgenic cell may be obtained by introducing the Cas13btransgene in an isolated cell. Delivery systems for transgenes are wellknown in the art. By means of example, the Cas13b transgene may bedelivered in for instance eukaryotic cell by means of vector (e.g., AAV,adenovirus, lentivirus) and/or particle and/or particle delivery, asalso described herein elsewhere.

It will be understood by the skilled person that the cell, such as theCas13b transgenic cell, as referred to herein may comprise furthergenomic alterations besides having an integrated Cas13b gene or themutations arising from the sequence specific action of Cas13b whencomplexed with RNA capable of guiding Cas13b to a target locus, such asfor instance one or more oncogenic mutations, as for instance andwithout limitation described in Platt et al. (2014), Chen et al., (2014)or Kumar et al. (2009).

In some embodiments, the Cas13b sequence is fused to one or more nuclearlocalization sequences (NLSs), such as about or more than about 1, 2, 3,4, 5, 6, 7, 8, 9, 10, or more NLSs. In some embodiments, the Cas13bcomprises about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore NLSs at or near the amino-terminus, about or more than about 1, 2,3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near the carboxy-terminus,or a combination of these (e.g. zero or at least one or more NLS at theamino-terminus and zero or at one or more NLS at the carboxy terminus).When more than one NLS is present, each may be selected independently ofthe others, such that a single NLS may be present in more than one copyand/or in combination with one or more other NLSs present in one or morecopies. In a preferred embodiment of the invention, the Cas13b comprisesat most 6 NLSs. In some embodiments, an NLS is considered near the N- orC-terminus when the nearest amino acid of the NLS is within about 1, 2,3, 4, 5, 10, 15, 20, 25, 30, 40, 50, or more amino acids along thepolypeptide chain from the N- or C-terminus. Non-limiting examples ofNLSs include an NLS sequence derived from: the NLS of the SV40 viruslarge T-antigen, having the amino acid sequence PKKKRKV(SEQ ID NO: 1);the NLS from nucleoplasmin (e.g. the nucleoplasmin bipartite NLS withthe sequence KRPAATKKAGQAKKKK) (SEQ ID NO: 2); the c-myc NLS having theamino acid sequence PAAKRVKLD (SEQ ID NO: 3) or RQRRNELKRSP(SEQ ID NO:4); the hRNPA1 M9 NLS having the sequenceNQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY(SEQ ID NO: 5); the sequenceRMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 6) of the IBBdomain from importin-alpha; the sequences VSRKRPRP (SEQ ID NO: 7) andPPKKARED (SEQ ID NO: 8) of the myoma T protein; the sequence PXPKKKPL(SEQ ID NO: 9) of human p53; the sequence SALIKKKKKMAP (SEQ ID NO: 10)of mouse c-abl IV; the sequences DRLRR (SEQ ID NO: 11) and PKQKKRK (SEQID NO: 12) of the influenza virus NS1; the sequence RKLKKKIKKL (SEQ IDNO: 13) of the Hepatitis virus delta antigen; the sequence REKKKFLKRR(SEQ ID NO:14) of the mouse Mx1 protein; the sequenceKRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 15) of the human poly(ADP-ribose)polymerase; and the sequence RKCLQAGMNLEARKTKK (SEQ ID NO: 16) of thesteroid hormone receptors (human) glucocorticoid. In general, the one ormore NLSs are of sufficient strength to drive accumulation of the Cas ina detectable amount in the nucleus of a eukaryotic cell. In general,strength of nuclear localization activity may derive from the number ofNLSs in the Cas, the particular NLS(s) used, or a combination of thesefactors. Detection of accumulation in the nucleus may be performed byany suitable technique. For example, a detectable marker may be fused tothe Cas, such that location within a cell may be visualized, such as incombination with a means for detecting the location of the nucleus (e.g.a stain specific for the nucleus such as DAPI). Cell nuclei may also beisolated from cells, the contents of which may then be analyzed by anysuitable process for detecting protein, such as immunohistochemistry,Western blot, or enzyme activity assay. Accumulation in the nucleus mayalso be determined indirectly, such as by an assay for the effect ofCRISPR complex formation (e.g. assay for DNA cleavage or mutation at thetarget sequence, or assay for altered gene expression activity affectedby CRISPR complex formation and/or Cas enzyme activity), as compared toa control no exposed to the Cas or complex, or exposed to a Cas lackingthe one or more NLSs.

In certain aspects the invention involves vectors, e.g. for deliveringor introducing in a cell Cas13b and/or RNA capable of guiding Cas13b toa target locus (i.e. guide RNA), but also for propagating thesecomponents (e.g. in prokaryotic cells). A used herein, a “vector” is atool that allows or facilitates the transfer of an entity from oneenvironment to another. It is a replicon, such as a plasmid, phage, orcosmid, into which another DNA segment may be inserted so as to bringabout the replication of the inserted segment. Generally, a vector iscapable of replication when associated with the proper control elements.In general, the term “vector” refers to a nucleic acid molecule capableof transporting another nucleic acid to which it has been linked.Vectors include, but are not limited to, nucleic acid molecules that aresingle-stranded, double-stranded, or partially double-stranded; nucleicacid molecules that comprise one or more free ends, no free ends (e.g.circular); nucleic acid molecules that comprise DNA, RNA, or both; andother varieties of polynucleotides known in the art. One type of vectoris a “plasmid,” which refers to a circular double stranded DNA loop intowhich additional DNA segments can be inserted, such as by standardmolecular cloning techniques. Another type of vector is a viral vector,wherein virally-derived DNA or RNA sequences are present in the vectorfor packaging into a virus (e.g. retroviruses, replication defectiveretroviruses, adenoviruses, replication defective adenoviruses, andadeno-associated viruses (AAVs)). Viral vectors also includepolynucleotides carried by a virus for transfection into a host cell.Certain vectors are capable of autonomous replication in a host cellinto which they are introduced (e.g. bacterial vectors having abacterial origin of replication and episomal mammalian vectors). Othervectors (e.g., non-episomal mammalian vectors) are integrated into thegenome of a host cell upon introduction into the host cell, and therebyare replicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively-linked. Such vectors are referred to herein as “expressionvectors.” Common expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids.

Recombinant expression vectors can comprise a nucleic acid of theinvention in a form suitable for expression of the nucleic acid in ahost cell, which means that the recombinant expression vectors includeone or more regulatory elements, which may be selected on the basis ofthe host cells to be used for expression, that is operatively-linked tothe nucleic acid sequence to be expressed. Within a recombinantexpression vector, “operably linked” is intended to mean that thenucleotide sequence of interest is linked to the regulatory element(s)in a manner that allows for expression of the nucleotide sequence (e.g.in an in vitro transcription/translation system or in a host cell whenthe vector is introduced into the host cell). With regards torecombination and cloning methods, mention is made of U.S. patentapplication Ser. No. 10/815,730, published Sep. 2, 2004 as US2004-0171156 A1, the contents of which are herein incorporated byreference in their entirety.

The vector(s) can include the regulatory element(s), e.g., promoter(s).The vector(s) can comprise Cas13b encoding sequence(s), and/or a single,but possibly also can comprise at least 2, 3 or 8 or 16 or 32 or 48 or50 guide RNA(s) (e.g., crRNAs) encoding sequences, such as 1-2, 1-3, 1-41-5, 3-6, 3-7, 3-8, 3-9, 3-10, 3-8, 3-16, 3-30, 3-32, 3-48, 3-50 RNA(s)(e.g., crRNAs). In a single vector there can be a promoter for each RNA(e.g., crRNA(s)), advantageously when there are up to about 16 RNA(s)(e.g., crRNA(s)s); and, when a single vector provides for more than 16RNA(s) (e.g., crRNA(s)s), one or more promoter(s) can drive expressionof more than one of the RNA(s) (e.g., crRNA(s)s), e.g., when there are32 RNA(s) (e.g., sgRNAs or crRNA(s)), each promoter can drive expressionof two RNA(s) (e.g., sgRNAs or crRNA(s)), and when there are 48 RNA(s)(e.g., sgRNAs or crRNA(s)), each promoter can drive expression of threeRNA(s) (e.g., sgRNAs or crRNA(s)). By simple arithmetic and wellestablished cloning protocols and the teachings in this disclosure oneskilled in the art can readily practice the invention as to the RNA(s),e.g., sgRNA(s) or crRNA(s) for a suitable exemplary vector such as AAV,and a suitable promoter such as the U6 promoter, e.g., U6-sgRNAs or-crRNA(s). For example, the packaging limit of AAV is ˜4.7 kb. Theskilled person can readily fit about 12-16, e.g., 13 U6-sgRNA orcrRNA(s) cassettes in a single vector. This can be assembled by anysuitable means, such as a golden gate strategy used for TALE assembly(http://www.genome-engineering.org/taleffectors/). The skilled personcan also use a tandem guide strategy to increase the number of U6-sgRNAsor -crRNA(s) by approximately 1.5 times, e.g., to increase from 12-16,e.g., 13 to approximately 18-24, e.g., about 19 U6-sgRNAs or -crRNA(s).Therefore, one skilled in the art can readily reach approximately 18-24,e.g., about 19 promoter-RNAs, e.g., U6-sgRNAs or -crRNA(s) in a singlevector, e.g., an AAV vector. A further means for increasing the numberof promoters and RNAs, e.g., sgRNA(s) or crRNA(s) in a vector is to usea single promoter (e.g., U6) to express an array of RNAs, e.g., sgRNAsor crRNA(s) separated by cleavable sequences. And an even further meansfor increasing the number of promoter-RNAs, e.g., sgRNAs or crRNA(s) ina vector, is to express an array of promoter-RNAs, e.g., sgRNAs orcrRNA(s) separated by cleavable sequences in the intron of a codingsequence or gene; and, in this instance it is advantageous to use apolymerase II promoter, which can have increased expression and enablethe transcription of long RNA in a tissue specific manner. (see, e.g.,nar.oxfordjournals.org/content/34/7/e53.short,www.nature.com/mt/journal/v16/n9/abs/mt2008144a.html). In anadvantageous embodiment, AAV may package U6 tandem sgRNA targeting up toabout 50 genes. Accordingly, from the knowledge in the art and theteachings in this disclosure the skilled person can readily make and usevector(s), e.g., a single vector, expressing multiple RNAs or guides orsgRNAs or crRNA(s) under the control or operatively or functionallylinked to one or more promoters-especially as to the numbers of RNAs orguides or sgRNAs or crRNA(s) discussed herein, without any undueexperimentation.

The guide RNA(s), e.g., sgRNA(s) or crRNA(s) encoding sequences and/orCas13b encoding sequences, can be functionally or operatively linked toregulatory element(s) and hence the regulatory element(s) driveexpression. The promoter(s) can be constitutive promoter(s) and/orconditional promoter(s) and/or inducible promoter(s) and/or tissuespecific promoter(s). The promoter can be selected from the groupconsisting of RNA polymerases, pol I, pol II, pol III, T7, U6, H1,retroviral Rous sarcoma virus (RSV) LTR promoter, the cytomegalovirus(CMV) promoter, the SV40 promoter, the dihydrofolate reductase promoter,the β-actin promoter, the phosphoglycerol kinase (PGK) promoter, and theEF1α promoter. An advantageous promoter is the promoter is U6.

In an aspect of the invention, novel RNA targeting systems also referredto as RNA- or RNA-targeting CRISPR systems of the present applicationare based on herein-identified Cas13b proteins which do not require thegeneration of customized proteins to target specific RNA sequences butrather a single enzyme can be programmed by a RNA molecule to recognizea specific RNA target, in other words the enzyme can be recruited to aspecific RNA target using said RNA molecule.

In some embodiments, one or more elements of a nucleic acid-targetingsystem is derived from a particular organism comprising an endogenousCRISPR RNA-targeting system. In certain embodiments, the CRISPRRNA-targeting system is found in Eubacterium and Ruminococcus. Incertain embodiments, the effector protein comprises targeted andcollateral ssRNA cleavage activity. In certain embodiments, the effectorprotein comprises dual HEPN domains. In certain embodiments, theeffector protein lacks a counterpart to the Helical-1 domain of Cas13a.In certain embodiments, the effector protein is smaller than previouslycharacterized class 2 CRISPR effectors, with a median size of 928 aa.This median size is 190 aa (17%) less than that of Cas13c, more than 200aa (18%) less than that of Cas13b, and more than 300 aa (26%) less thanthat of Cas13a. In certain embodiments, the effector protein has norequirement for a flanking sequence (e.g., PFS, PAM).

In certain embodiments, the effector protein locus structures include aWYL domain containing accessory protein (so denoted after three aminoacids that were conserved in the originally identified group of thesedomains; see, e.g., WYL domain IPR026881). In certain embodiments, theWYL domain accessory protein comprises at least one helix-turn-helix(HTH) or ribbon-helix-helix (RHIH) DNA-binding domain. In certainembodiments, the WYL domain containing accessory protein increases boththe targeted and the collateral ssRNA cleavage activity of theRNA-targeting effector protein. In certain embodiments, the WYL domaincontaining accessory protein comprises an N-terminal RHH domain, as wellas a pattern of primarily hydrophobic conserved residues, including aninvariant tyrosine-leucine doublet corresponding to the original WYLmotif. In certain embodiments, the WYL domain containing accessoryprotein is WYL1. WYL1 is a single WYL-domain protein associatedprimarily with Ruminococcus.

In other example embodiments, the Type VI RNA-targeting Cas enzyme isCas 13d. In certain embodiments, Cas13d is Eubacterium siraeum DSM 15702(EsCas13d) or Ruminococcus sp. N15.MGS-57 (RspCas13d) (see, e.g., Yan etal., Cas13d Is a Compact RNA-Targeting Type VI CRISPR EffectorPositively Modulated by a WYL-Domain-Containing Accessory Protein,Molecular Cell (2018), doi.org/10.1016/j.molcel.2018.02.028). RspCas13dand EsCas13d have no flanking sequence requirements (e.g., PFS, PAM).

The nucleic acids-targeting systems, the vector systems, the vectors andthe compositions described herein may be used in various nucleicacids-targeting applications, altering or modifying synthesis of a geneproduct, such as a protein, nucleic acids cleavage, nucleic acidsediting, nucleic acids splicing; trafficking of target nucleic acids,tracing of target nucleic acids, isolation of target nucleic acids,visualization of target nucleic acids, etc.

In an advantageous embodiment, the present invention encompasses Cas13beffector proteins with reference to Table 1A or Table 1B. A Table 1A orTable 1B Cas13b effector protein is as discussed in more detail hereinin conjunction with Table 1A or Table 1B.

Cas13b Nucleases

The Cas13b effector protein of the invention is, or in, or comprises, orconsists essentially of, or consists of, or involves or relates to sucha protein from or as set forth in Table 1A or Table 1B. This inventionis intended to provide, or relate to, or involve, or comprise, orconsist essentially of, or consist of, a protein from or as set forth inTable 1A or 1B, including mutations or alterations thereof as set forthherein A Table 1A or Table 1B Cas13b effector protein is as discussed inmore detail herein in conjunction with Table 1A or Table 1B.

Thus, in some embodiments, the effector protein may be a RNA-bindingprotein, such as a dead-Cas type effector protein, which may beoptionally functionalised as described herein for instance with antranscriptional activator or repressor domain, NLS or other functionaldomain. In some embodiments, the effector protein may be a RNA-bindingprotein that cleaves a single strand of RNA. If the RNA bound is ssRNA,then the ssRNA is fully cleaved. In some embodiments, the effectorprotein may be a RNA-binding protein that cleaves a double strand ofRNA, for example if it comprises two RNase domains. If the RNA bound isdsRNA, then the dsRNA is fully cleaved. In some embodiments, theeffector protein may be a RNA-binding protein that has nickase activity,i.e. it binds dsRNA, but only cleaves one of the RNA strands.

RNase function in CRISPR systems is known, for example mRNA targetinghas been reported for certain type III CRISPR-Cas systems (Hale et al.,2014, Genes Dev, vol. 28, 2432 2443; Hale et al., 2009, Cell, vol. 139,945-956; Peng et al., 2015, Nucleic acids research, vol. 43, 406-417)and provides significant advantages. A CRISPR-Cas system, composition ormethod targeting RNA via the present effector proteins is thus provided.

The target RNA, i.e. the RNA of interest, is the RNA to be targeted bythe present invention leading to the recruitment to, and the binding ofthe effector protein at, the target site of interest on the target RNA.The target RNA may be any suitable form of RNA. This may include, insome embodiments, mRNA. In other embodiments, the target RNA may includetRNA or rRNA.

Interfering RNA (RNAi) and microRNA (miRNA)

In other embodiments, the target RNA may include interfering RNA, i.e.RNA involved in an RNA interference pathway, such as shRNA, siRNA and soforth. In other embodiments, the target RNA may include microRNA(miRNA). Control over interfering RNA or miRNA may help reduceoff-target effects (OTE) seen with those approaches by reducing thelongevity of the interfering RNA or miRNA in vivo or in vitro.

If the effector protein and suitable guide are selectively expressed(for example spatially or temporally under the control of a suitablepromoter, for example a tissue- or cell cycle-specific promoter and/orenhancer) then this could be used to ‘protect’ the cells or systems (invivo or in vitro) from RNAi in those cells. This may be useful inneighbouring tissues or cells where RNAi is not required or for thepurposes of comparison of the cells or tissues where the effectorprotein and suitable guide are and are not expressed (i.e. where theRNAi is not controlled and where it is, respectively). The effectorprotein may be used to control or bind to molecules comprising orconsisting of RNA, such as ribozymes, ribosomes or riboswitches. Inembodiments of the invention, the RNA guide can recruit the effectorprotein to these molecules so that the effector protein is able to bindto them.

Ribosomal RNA (rRNA)

For example, azalide antibiotics such as azithromycin, are well known.They target and disrupt the 50S ribosomal subunit. The present effectorprotein, together with a suitable guide RNA to target the 50S ribosomalsubunit, may be, in some embodiments, recruited to and bind to the 50Sribosomal subunit. Thus, the present effector protein in concert with asuitable guide directed at a ribosomal (especially the 50s ribosomalsubunit) target is provided. Use of this use effector protein in concertwith the suitable guide directed at the ribosomal (especially the 50sribosomal subunit) target may include antibiotic use. In particular, theantibiotic use is analogous to the action of azalide antibiotics, suchas azithromycin. In some embodiments, prokaryotic ribosomal subunits,such as the 70S subunit in prokaryotes, the 50S subunit mentioned above,the 30S subunit, as well as the 16S and 5S subunits may be targeted. Inother embodiments, eukaryotic ribosomal subunits, such as the 80Ssubunit in eukaryotes, the 60S subunit, the 40S subunit, as well as the28S, 18S. 5.8S and 5S subunits may be targeted.

The effector protein may be a RNA-binding protein, optionallyfunctionalised, as described herein. In some embodiments, the effectorprotein may be a RNA-binding protein that cleaves a single strand ofRNA. In either case, but particularly where the RNA-binding proteincleaves a single strand of RNA, then ribosomal function may be modulatedand, in particular, reduced or destroyed. This may apply to anyribosomal RNA and any ribosomal subunit and the sequences of rRNA arewell known.

Control of ribosomal activity is thus envisaged through use of thepresent effector protein in concert with a suitable guide to theribosomal target. This may be through cleavage of, or binding to, theribosome. In particular, reduction of ribosomal activity is envisaged.This may be useful in assaying ribosomal function in vivo or in vitro,but also as a means of controlling therapies based on ribosomalactivity, in vivo or in vitro. Furthermore, control (i.e. reduction) ofprotein synthesis in an in vivo or in vitro system is envisaged, suchcontrol including antibiotic and research and diagnostic use.

Riboswitches

A riboswitch (also known as an aptozyme) is a regulatory segment of amessenger RNA molecule that binds a small molecule. This typicallyresults in a change in production of the proteins encoded by the mRNA.Thus, control of riboswitch activity is thus envisaged through use ofthe present effector protein in concert with a suitable guide to theriboswitch target. This may be through cleavage of, or binding to, theriboswitch. In particular, reduction of riboswitch activity isenvisaged. This may be useful in assaying riboswitch function in vivo orin vitro, but also as a means of controlling therapies based onriboswitch activity, in vivo or in vitro. Furthermore, control (i.e.reduction) of protein synthesis in an in vivo or in vitro system isenvisaged. This control, as for rRNA may include antibiotic and researchand diagnostic use.

Ribozymes

Ribozymes are RNA molecules having catalytic properties, analogous toenzymes (which are of course proteins). As ribozymes, both naturallyoccurring and engineered, comprise or consist of RNA, they may also betargeted by the present RNA-binding effector protein. In someembodiments, the effector protein may be a RNA-binding protein cleavesthe ribozyme to thereby disable it. Control of ribozymal activity isthus envisaged through use of the present effector protein in concertwith a suitable guide to the ribozymal target. This may be throughcleavage of, or binding to, the ribozyme. In particular, reduction ofribozymal activity is envisaged. This may be useful in assayingribozymal function in vivo or in vitro, but also as a means ofcontrolling therapies based on ribozymal activity, in vivo or in vitro.

Gene Expression, Including RNA Processing

The effector protein may also be used, together with a suitable guide,to target gene expression, including via control of RNA processing. Thecontrol of RNA processing may include RNA processing reactions such asRNA splicing, including alternative splicing, via targeting of RNApol;viral replication (in particular of satellite viruses, bacteriophagesand retroviruses, such as HBV, HBC and HIV and others listed herein)including viroids in plants; and tRNA biosynthesis. The effector proteinand suitable guide may also be used to control RNAactivation (RNAa).RNAa leads to the promotion of gene expression, so control of geneexpression may be achieved that way through disruption or reduction ofRNAa and thus less promotion of gene expression.

RNAi Screens

Identifying gene products whose knockdown is associated with phenotypicchanges, biological pathways can be interrogated and the constituentparts identified, via RNAi screens. Control may also be exerted over orduring these screens by use of the effector protein and suitable guideto remove or reduce the activity of the RNAi in the screen and thusreinstate the activity of the (previously interfered with) gene product(by removing or reducing the interference/repression).

Satellite RNAs (satRNAs) and satellite viruses may also be treated.

Control herein with reference to RNase activity generally meansreduction, negative disruption or known-down or knock out.

In Vivo RNA Applications Inhibition of Gene Expression

The target-specific RNAses provided herein allow for very specificcutting of a target RNA. The interference at RNA level allows formodulation both spatially and temporally and in a non-invasive way, asthe genome is not modified.

A number of diseases have been demonstrated to be treatable by mRNAtargeting. While most of these studies relate to administration ofsiRNA, it is clear that the RNA targeting effector proteins providedherein can be applied in a similar way.

Examples of mRNA targets (and corresponding disease treatments) areVEGF, VEGF-R1 and RTP801 (in the treatment of AMD and/or DME), Caspase 2(in the treatment of Naion)ADRB2 (in the treatment of intraocularpressure), TRPVI (in the treatment of Dry eye syndrome, Syk kinase (inthe treatment of asthma), Apo B (in the treatment ofhypercholesterolemia), PLK1, KSP and VEGF (in the treatment of solidtumors), Ber-Abl (in the treatment of CML) (Burnett and Rossi Chem Biol.2012, 19(1): 60-71)). Similarly, RNA targeting has been demonstrated tobe effective in the treatment of RNA-virus mediated diseases such as HIV(targeting of HIV Tet and Rev), RSV (targeting of RSV nucleocapsid) andHCV (targeting of miR-122) (Burnett and Rossi Chem Biol. 2012, 19(1):60-71).

It is further envisaged that the RNA targeting effector protein of theinvention can be used for mutation specific or allele specificknockdown. Guide RNA's can be designed that specifically target asequence in the transcribed mRNA comprising a mutation or anallele-specific sequence. Such specific knockdown is particularlysuitable for therapeutic applications relating to disorders associatedwith mutated or allele-specific gene products. For example, most casesof familial hypobetalipoproteinemia (FHBL) are caused by mutations inthe ApoB gene. This gene encodes two versions of the apolipoprotein Bprotein: a short version (ApoB-48) and a longer version (ApoB-100).Several ApoB gene mutations that lead to FHBL cause both versions ofApoB to be abnormally short. Specifically targeting and knockdown ofmutated ApoB mRNA transcripts with an RNA targeting effector protein ofthe invention may be beneficial in treatment of FHBL. As anotherexample, Huntington's disease (HD) is caused by an expansion of CAGtriplet repeats in the gene coding for the Huntingtin protein, whichresults in an abnormal protein. Specifically targeting and knockdown ofmutated or allele-specific mRNA transcripts encoding the Huntingtinprotein with an RNA targeting effector protein of the invention may bebeneficial in treatment of HD.

It is noted that in this context, and more generally for the variousapplications as described herein, the use of a split version of the RNAtargeting effector protein can be envisaged. Indeed, this may not onlyallow increased specificity but may also be advantageous for delivery.The Cas13b is split in the sense that the two parts of the Cas13b enzymesubstantially comprise a functioning Cas13b. Ideally, the split shouldalways be so that the catalytic domain(s) are unaffected. That Cas13bmay function as a nuclease or it may be a dead-Cas13b which isessentially an RNA-binding protein with very little or no catalyticactivity, due to typically mutation(s) in its catalytic domains.

Each half of the split Cas13b may be fused to a dimerization partner. Bymeans of example, and without limitation, employing rapamycin sensitivedimerization domains, allows to generate a chemically inducible splitCas13b for temporal control of Cas13b activity. Cas13b can thus berendered chemically inducible by being split into two fragments and thatrapamycin-sensitive dimerization domains may be used for controlledreassembly of the Cas13b. The two parts of the split Cas13b can bethought of as the N′ terminal part and the C′ terminal part of the splitCas13b. The fusion is typically at the split point of the Cas13b. Inother words, the C′ terminal of the N′ terminal part of the split Cas13bis fused to one of the dimer halves, whilst the N′ terminal of the C′terminal part is fused to the other dimer half.

The Cas13b does not have to be split in the sense that the break isnewly created. The split point is typically designed in silico andcloned into the constructs. Together, the two parts of the split Cas13b,the N′ terminal and C′ terminal parts, form a full Cas13b, comprisingpreferably at least 70% or more of the wildtype amino acids (ornucleotides encoding them), preferably at least 80% or more, preferablyat least 90% or more, preferably at least 95% or more, and mostpreferably at least 99% or more of the wildtype amino acids (ornucleotides encoding them). Some trimming may be possible, and mutantsare envisaged. Non-functional domains may be removed entirely. What isimportant is that the two parts may be brought together and that thedesired Cas13b function is restored or reconstituted. The dimer may be ahomodimer or a heterodimer.

In certain embodiments, the Cas13b effector as described herein may beused for mutation-specific, or allele-specific targeting, such as formutation-specific, or allele-specific knockdown.

The RNA targeting effector protein can moreover be fused to anotherfunctional RNAse domain, such as a non-specific RNase or Argonaute 2,which acts in synergy to increase the RNAse activity or to ensurefurther degradation of the message.

Modulation of Gene Expression Through Modulation of RNA Function

Apart from a direct effect on gene expression through cleavage of themRNA, RNA targeting can also be used to impact specific aspects of theRNA processing within the cell, which may allow a more subtle modulationof gene expression. Generally, modulation can for instance be mediatedby interfering with binding of proteins to the RNA, such as for instanceblocking binding of proteins, or recruiting RNA binding proteins.Indeed, modulations can be ensured at different levels such as splicing,transport, localization, translation and turnover of the mRNA. Similarlyin the context of therapy, it can be envisaged to address (pathogenic)malfunctioning at each of these levels by using RNA-specific targetingmolecules. In these embodiments it is in many cases preferred that theRNA targeting protein is a “dead” Cas13b that has lost the ability tocut the RNA target but maintains its ability to bind thereto, such asthe mutated forms of Cas13b described herein.

a) Alternative Splicing

Many of the human genes express multiple mRNAs as a result ofalternative splicing. Different diseases have been shown to be linked toaberrant splicing leading to loss of function or gain of function of theexpressed gene. While some of these diseases are caused by mutationsthat cause splicing defects, a number of these are not. One therapeuticoption is to target the splicing mechanism directly. The RNA targetingeffector proteins described herein can for instance be used to block orpromote slicing, include or exclude exons and influence the expressionof specific isoforms and/or stimulate the expression of alternativeprotein products. Such applications are described in more detail below.

A RNA targeting effector protein binding to a target RNA can stericallyblock access of splicing factors to the RNA sequence. The RNA targetingeffector protein targeted to a splice site may block splicing at thesite, optionally redirecting splicing to an adjacent site. For instancea RNA targeting effector protein binding to the 5′ splice site bindingcan block the recruitment of the U1 component of the spliceosome,favoring the skipping of that exon. Alternatively, a RNA targetingeffector protein targeted to a splicing enhancer or silencer can preventbinding of transacting regulatory splicing factors at the target siteand effectively block or promote splicing. Exon exclusion can further beachieved by recruitment of ILF2/3 to precursor mRNA near an exon by anRNA targeting effector protein as described herein. As yet anotherexample, a glycine rich domain can be attached for recruitment of hnRNPA1 and exon exclusion (Del Gatto-Konczak et al. Mol Cell Biol. 1999Jan.; 19(1):251-60).

In certain embodiments, through appropriate selection of gRNA, specificsplice variants may be targeted, while other splice variants will not betargeted.

In some cases the RNA targeting effector protein can be used to promoteslicing (e.g. where splicing is defective). For instance a RNA targetingeffector protein can be associated with an effector capable ofstabilizing a splicing regulatory stem-loop in order to furthersplicing. The RNA targeting effector protein can be linked to aconsensus binding site sequence for a specific splicing factor in orderto recruit the protein to the target DNA.

Examples of diseases which have been associated with aberrant splicinginclude, but are not limited to Paraneoplastic Opsoclonus MyoclonusAtaxia (or POMA), resulting from a loss of Nova proteins which regulatesplicing of proteins that function in the synapse, and Cystic Fibrosis,which is caused by defective splicing of a cystic fibrosis transmembraneconductance regulator, resulting in the production of nonfunctionalchloride channels. In other diseases aberrant RNA splicing results ingain-of-function. This is the case for instance in myotonic dystrophywhich is caused by a CUG triplet-repeat expansion (from 50 to >1500repeats) in the 3′UTR of an mRNA, causing splicing defects.

The RNA targeting effector protein can be used to include an exon byrecruiting a splicing factor (such as U1) to a 5′splicing site topromote excision of introns around a desired exon. Such recruitmentcould be mediated trough a fusion with an arginine/serine rich domain,which functions as splicing activator (Gravely B R and Maniatis T, MolCell. 1998 (5):765-71).

It is envisaged that the RNA targeting effector protein can be used toblock the splicing machinery at a desired locus, resulting in preventingexon recognition and the expression of a different protein product. Anexample of a disorder that may treated is Duchenne muscular dystrophy(DMD), which is caused by mutations in the gene encoding for thedystrophin protein. Almost all DMD mutations lead to frameshifts,resulting in impaired dystrophin translation. The RNA targeting effectorprotein can be paired with splice junctions or exonic splicing enhancers(ESEs) thereby preventing exon recognition, resulting in the translationof a partially functional protein. This converts the lethal Duchennephenotype into the less severe Becker phenotype.

b) RNA Modification

RNA editing is a natural process whereby the diversity of gene productsof a given sequence is increased by minor modification in the RNA.Typically, the modification involves the conversion of adenosine (A) toinosine (I), resulting in an RNA sequence which is different from thatencoded by the genome. RNA modification is generally ensured by the ADARenzyme, whereby the pre-RNA target forms an imperfect duplex RNA bybase-pairing between the exon that contains the adenosine to be editedand an intronic non-coding element. A classic example of A-I editing isthe glutamate receptor GluR-B mRNA, whereby the change results inmodified conductance properties of the channel (Higuchi M, et al. Cell.1993; 75:1361-70).

In humans, a heterozygous functional-null mutation in the ADAR1 geneleads to a skin disease, human pigmentary genodermatosis (Miyamura Y, etal. Am J Hum Genet. 2003; 73:693 9). It is envisaged that the RNAtargeting effector proteins of the present invention can be used tocorrect malfunctioning RNA modification.

It is further envisaged that RNA adenosine methylase(N(6)-methyladenosine) can be fused to the RNA targeting effectorproteins of the invention and targeted to a transcript of interest. Thismethylase causes reversible methylation, has regulatory roles and mayaffect gene expression and cell fate decisions by modulating multipleRNA-related cellular pathways (Fu et al Nat Rev Genet. 2014;15(5):293-306).

c) Polyadenylation

Polyadenylation of an mRNA is important for nuclear transport,translation efficiency and stability of the mRNA, and all of these, aswell as the process of polyadenylation, depend on specific RBPs. Mosteukaryotic mRNAs receive a 3′ poly(A) tail of about 200 nucleotidesafter transcription. Polyadenylation involves different RNA-bindingprotein complexes which stimulate the activity of a poly(A)polymerase(Minvielle-Sebastia L et al. Curr Opin Cell Biol. 1999; 11:352 7). It isenvisaged that the RNA-targeting effector proteins provided herein canbe used to interfere with or promote the interaction between theRNA-binding proteins and RNA.

Examples of diseases which have been linked to defective proteinsinvolved in polyadenylation are oculopharyngeal muscular dystrophy(OPMD) (Brais B, et al. Nat Genet. 1998; 18:164-7).

d) RNA Export

After pre-mRNA processing, the mRNA is exported from the nucleus to thecytoplasm. This is ensured by a cellular mechanism which involves thegeneration of a carrier complex, which is then translocated through thenuclear pore and releases the mRNA in the cytoplasm, with subsequentrecycling of the carrier.

Overexpression of proteins (such as TAP) which play a role in the exportof RNA has been found to increase export of transcripts that areotherwise ineffeciently exported in Xenopus (Katahira J, et al. EMBO J.1999; 18:2593-609).

e) mRNA Localization

mRNA localization ensures spatially regulated protein production.Localization of transcripts to a specific region of the cell can beensured by localization elements. In particular embodiments, it isenvisaged that the effector proteins described herein can be used totarget localization elements to the RNA of interest. The effectorproteins can be designed to bind the target transcript and shuttle themto a location in the cell determined by its peptide signal tag. Moreparticularly for instance, a RNA targeting effector protein fused to anuclear localization signal (NLS) can be used to alter RNA localization.

Further examples of localization signals include the zipcode bindingprotein (ZBP1) which ensures localization of β-actin to the cytoplasm inseveral asymmetric cell types, KDEL retention sequence (localization toendoplasmic reticulum), nuclear export signal (localization tocytoplasm), mitochondrial targeting signal (localization tomitochondria), peroxisomal targeting signal (localization to peroxisome)and m6A marking/YTHDF2 (localization to p-bodies). Other approaches thatare envisaged are fusion of the RNA targeting effector protein withproteins of known localization (for instance membrane, synapse).

Alternatively, the effector protein according to the invention may forinstance be used in localization-dependent knockdown. By fusing theeffector protein to a appropriate localization signal, the effector istargeted to a particular cellular compartment. Only target RNAs residingin this compartment will effectively be targeted, whereas otherwiseidentical targets, but residing in a different cellular compartment willnot be targeted, such that a localization dependent knockdown can beestablished.

f) Translation

The RNA targeting effector proteins described herein can be used toenhance or repress translation. It is envisaged that upregulatingtranslation is a very robust way to control cellular circuits. Further,for functional studies a protein translation screen can be favorableover transcriptional upregulation screens, which have the shortcomingthat upregulation of transcript does not translate into increasedprotein production.

It is envisaged that the RNA targeting effector proteins describedherein can be used to bring translation initiation factors, such asEIF4G in the vicinity of the 5′ untranslated repeat (5′UTR) of amessenger RNA of interest to drive translation (as described in DeGregorio et al. EMBO J. 1999; 18(17):4865-74 for a non-reprogrammableRNA binding protein). As another example GLD2, a cytoplasmic poly(A)polymerase, can be recruited to the target mRNA by an RNA targetingeffector protein. This would allow for directed polyadenylation of thetarget mRNA thereby stimulating translation.

Similarly, the RNA targeting effector proteins envisaged herein can beused to block translational repressors of mRNA, such as ZBP1(Huttelmaier S, et al. Nature. 2005; 438:512-5). By binding totranslation initiation site of a target RNA, translation can be directlyaffected.

In addition, fusing the RNA targeting effector proteins to a proteinthat stabilizes mRNAs, e.g. by preventing degradation thereof such asRNase inhibitors, it is possible to increase protein production from thetranscripts of interest.

It is envisaged that the RNA targeting effector proteins describedherein can be used to repress translation by binding in the 5′ UTRregions of a RNA transcript and preventing the ribosome from forming andbeginning translation.

Further, the RNA targeting effector protein can be used to recruit Caf1,a component of the CCR4-NOT deadenylase complex, to the target mRNA,resulting in deadenylation or the target transcript and inhibition ofprotein translation.

For instance, the RNA targeting effector protein of the invention can beused to increase or decrease translation of therapeutically relevantproteins. Examples of therapeutic applications wherein the RNA targetingeffector protein can be used to downregulate or upregulate translationare in amyotrophic lateral sclerosis (ALS) and cardiovascular disorders.Reduced levels of the glial glutamate transporter EAAT2 have beenreported in ALS motor cortex and spinal cord, as well as multipleabnormal EAAT2 mRNA transcripts in ALS brain tissue. Loss of the EAAT2protein and function thought to be the main cause of excitotoxicity inALS. Restoration of EAAT2 protein levels and function may providetherapeutic benefit. Hence, the RNA targeting effector protein can bebeneficially used to upregulate the expression of EAAT2 protein, e.g. byblocking translational repressors or stabilizing mRNA as describedabove. Apolipoprotein A1 is the major protein component of high densitylipoprotein (HDL) and ApoA1 and HDL are generally considered asatheroprotective. It is envisages that the RNA targeting effectorprotein can be beneficially used to upregulate the expression of ApoA1,e.g. by blocking translational repressors or stabilizing mRNA asdescribed above.

g) mRNA Turnover

Translation is tightly coupled to mRNA turnover and regulated mRNAstability. Specific proteins have been described to be involved in thestability of transcripts (such as the ELAV/Hu proteins in neurons, KeeneJ D, 1999, Proc Natl Acad Sci USA. 96:5-7) and tristetraprolin (TTP).These proteins stabilize target mRNAs by protecting the messages fromdegradation in the cytoplasm (Peng S S et al., 1988, EMBO J.17:3461-70).

It can be envisaged that the RNA-targeting effector proteins of thepresent invention can be used to interfere with or to promote theactivity of proteins acting to stabilize mRNA transcripts, such thatmRNA turnover is affected. For instance, recruitment of human TTP to thetarget RNA using the RNA targeting effector protein would allow foradenylate-uridylate-rich element (AU-rich element) mediatedtranslational repression and target degradation. AU-rich elements arefound in the 3′ UTR of many mRNAs that code for proto-oncogenes, nucleartranscription factors, and cytokines and promote RNA stability. Asanother example, the RNA targeting effector protein can be fused to HuR,another mRNA stabilization protein (Hinman M N and Lou H, Cell Mol LifeSci 2008; 65:3168-81), and recruit it to a target transcript to prolongits lifetime or stabilize short-lived mRNA.

It is further envisaged that the RNA-targeting effector proteinsdescribed herein can be used to promote degradation of targettranscripts. For instance, m6A methyltransferase can be recruited to thetarget transcript to localize the transcript to P-bodies leading todegradation of the target.

As yet another example, an RNA targeting effector protein as describedherein can be fused to the non-specific endonuclease domain PilTN-terminus (PIN), to recruit it to a target transcript and allowdegradation thereof.

Patients with paraneoplastic neurological disorder (PND)-associatedencephalomyelitis and neuropathy are patients who develop autoantibodiesagainst Hu-proteins in tumors outside of the central nervous system(Szabo A et al. 1991, Cell; 67:325-33 which then cross the blood-brainbarrier. It can be envisaged that the RNA-targeting effector proteins ofthe present invention can be used to interfere with the binding ofauto-antibodies to mRNA transcripts.

Patients with dystrophy type 1 (DM1), caused by the expansion of (CUG)nin the 3′ UTR of dystrophia myotonica-protein kinase (DMPK) gene, arecharacterized by the accumulation of such transcripts in the nucleus. Itis envisaged that the RNA targeting effector proteins of the inventionfused with an endonuclease targeted to the (CUG)n repeats could inhibitsuch accumulation of aberrant transcripts.

h) Interaction with Multi Functional Proteins

Some RNA-binding proteins bind to multiple sites on numerous RNAs tofunction in diverse processes. For instance, the hnRNP A1 protein hasbeen found to bind exonic splicing silencer sequences, antagonizing thesplicing factors, associate with telomere ends (thereby stimulatingtelomere activity) and bind miRNA to facilitate Drosha-mediatedprocessing thereby affecting maturation. It is envisaged that theRNA-binding effector proteins of the present invention can interferewith the binding of RNA-binding proteins at one or more locations.

i) RNA Folding

RNA adopts a defined structure in order to perform its biologicalactivities. Transitions in conformation among alternative tertiarystructures are critical to most RNA-mediated processes. However, RNAfolding can be associated with several problems. For instance, RNA mayhave a tendency to fold into, and be upheld in, improper alternativeconformations and/or the correct tertiary structure may not besufficiently thermodynamically favored over alternative structures. TheRNA targeting effector protein, in particular a cleavage-deficient ordead RNA targeting protein, of the invention may be used to directfolding of (m)RNA and/or capture the correct tertiary structure thereof.

Use of RNA-Targeting Effector Protein in Modulating Cellular Status

In certain embodiments Cas13b in a complex with crRNA is activated uponbinding to target RNA and subsequently cleaves any nearby ssRNA targets(i.e. “collateral” or “bystander” effects). Cas13b, once primed by thecognate target, can cleave other (non-complementary) RNA molecules. Suchpromiscuous RNA cleavage could potentially cause cellular toxicity, orotherwise affect cellular physiology or cell status.

Accordingly, in certain embodiments, the non-naturally occurring orengineered composition, vector system, or delivery systems as describedherein are used for or are for use in induction of cell dormancy. Incertain embodiments, the non-naturally occurring or engineeredcomposition, vector system, or delivery systems as described herein areused for or are for use in induction of cell cycle arrest. In certainembodiments, the non-naturally occurring or engineered composition,vector system, or delivery systems as described herein are used for orare for use in reduction of cell growth and/or cell proliferation. Incertain embodiments, the non-naturally occurring or engineeredcomposition, vector system, or delivery systems as described herein areused for or are for use in induction of cell anergy. In certainembodiments, the non-naturally occurring or engineered composition,vector system, or delivery systems as described herein are used for orare for use in induction of cell apoptosis. In certain embodiments, thenon-naturally occurring or engineered composition, vector system, ordelivery systems as described herein are used for or are for use inincuction of cell necrosis. In certain embodiments, the non-naturallyoccurring or engineered composition, vector system, or delivery systemsas described herein are used for or are for use in induction of celldeath. In certain embodiments, the non-naturally occurring or engineeredcomposition, vector system, or delivery systems as described herein areused for or are for use in induction of programmed cell death.

In certain embodiments, the invention relates to a method for inductionof cell dormancy comprising introducing or inducing the non-naturallyoccurring or engineered composition, vector system, or delivery systemsas described herein. In certain embodiments, the invention relates to amethod for induction of cell cycle arrest comprising introducing orinducing the non-naturally occurring or engineered composition, vectorsystem, or delivery systems as described herein. In certain embodiments,the invention relates to a method for reduction of cell growth and/orcell proliferation comprising introducing or inducing the non-naturallyoccurring or engineered composition, vector system, or delivery systemsas described herein. In certain embodiments, the invention relates to amethod for induction of cell anergy comprising introducing or inducingthe non-naturally occurring or engineered composition, vector system, ordelivery systems as described herein. In certain embodiments, theinvention relates to a method for induction of cell apoptosis comprisingintroducing or inducing the non-naturally occurring or engineeredcomposition, vector system, or delivery systems as described herein. Incertain embodiments, the invention relates to a method for induction ofcell necrosis comprising introducing or inducing the non-naturallyoccurring or engineered composition, vector system, or delivery systemsas described herein. In certain embodiments, the invention relates to amethod for induction of cell death comprising introducing or inducingthe non-naturally occurring or engineered composition, vector system, ordelivery systems as described herein. In certain embodiments, theinvention relates to a method for induction of programmed cell deathcomprising introducing or inducing the non-naturally occurring orengineered composition, vector system, or delivery systems as describedherein.

The methods and uses as described herein may be therapeutic orprophylactic and may target particular cells, cell (sub)populations, orcell/tissue types. In particular, the methods and uses as describedherein may be therapeutic or prophylactic and may target particularcells, cell (sub)populations, or cell/tissue types expressing one ormore target sequences, such as one or more particular target RNA (e.g.ss RNA). Without limitation, target cells may for instance be cancercells expressing a particular transcript, e.g. neurons of a given class,(immune) cells causing e.g. autoimmunity, or cells infected by aspecific (e.g. viral) pathogen, etc.

Accordingly, in certain embodiments, the invention relates to a methodfor treating a pathological condition characterized by the presence ofundesirable cells (host cells), comprising introducing or inducing thenon-naturally occurring or engineered composition, vector system, ordelivery systems as described herein. In certain embodiments, theinvention relates the use of the non-naturally occurring or engineeredcomposition, vector system, or delivery systems as described herein fortreating a pathological condition characterized by the presence ofundesirable cells (host cells). In certain embodiments, the inventionrelates the non-naturally occurring or engineered composition, vectorsystem, or delivery systems as described herein for use in treating apathological condition characterized by the presence of undesirablecells (host cells). It is to be understood that preferably theCRISPR-Cas system targets a target specific for the undesirable cells.In certain embodiments, the invention relates to the use of thenon-naturally occurring or engineered composition, vector system, ordelivery systems as described herein for treating, preventing, oralleviating cancer. In certain embodiments, the invention relates to thenon-naturally occurring or engineered composition, vector system, ordelivery systems as described herein for use in treating, preventing, oralleviating cancer. In certain embodiments, the invention relates to amethod for treating, preventing, or alleviating cancer comprisingintroducing or inducing the non-naturally occurring or engineeredcomposition, vector system, or delivery systems as described herein. Itis to be understood that preferably the CRISPR-Cas system targets atarget specific for the cancer cells. In certain embodiments, theinvention relates to the use of the non-naturally occurring orengineered composition, vector system, or delivery systems as describedherein for treating, preventing, or alleviating infection of cells by apathogen. In certain embodiments, the invention relates to thenon-naturally occurring or engineered composition, vector system, ordelivery systems as described herein for use in treating, preventing, oralleviating infection of cells by a pathogen. In certain embodiments,the invention relates to a method for treating, preventing, oralleviating infection of cells by a pathogen comprising introducing orinducing the non-naturally occurring or engineered composition, vectorsystem, or delivery systems as described herein. It is to be understoodthat preferably the CRISPR-Cas system targets a target specific for thecells infected by the pathogen (e.g. a pathogen derived target). Incertain embodiments, the invention relates to the use of thenon-naturally occurring or engineered composition, vector system, ordelivery systems as described herein for treating, preventing, oralleviating an autoimmune disorder. In certain embodiments, theinvention relates to the non-naturally occurring or engineeredcomposition, vector system, or delivery systems as described herein foruse in treating, preventing, or alleviating an autoimmune disorder. Incertain embodiments, the invention relates to a method for treating,preventing, or alleviating an autoimmune disorder comprising introducingor inducing the non-naturally occurring or engineered composition,vector system, or delivery systems as described herein. It is to beunderstood that preferably the CRISPR-Cas system targets a targetspecific for the cells responsible for the autoimmune disorder (e.g.specific immune cells).

Use of RNA-Targeting Effector Protein in RNA Detection

It is further envisaged that the RNA targeting effector protein can beused in Northern blot assays. Northern blotting involves the use ofelectrophoresis to separate RNA samples by size. The RNA targetingeffector protein can be used to specifically bind and detect the targetRNA sequence.

A RNA targeting effector protein can be fused to a fluorescent protein(such as GFP) and used to track RNA localization in living cells. Moreparticularly, the RNA targeting effector protein can be inactivated inthat it no longer cleaves RNA. In particular embodiments, it isenvisaged that a split RNA targeting effector protein can be used,whereby the signal is dependent on the binding of both subproteins, inorder to ensure a more precise visualization. Alternatively, a splitfluorescent protein can be used that is reconstituted when multiple RNAtargeting effector protein complexes bind to the target transcript. Itis further envisaged that a transcript is targeted at multiple bindingsites along the mRNA so the fluorescent signal can amplify the truesignal and allow for focal identification. As yet another alternative,the fluorescent protein can be reconstituted form a split intein.

RNA targeting effector proteins are for instance suitably used todetermine the localization of the RNA or specific splice variants, thelevel of mRNA transcript, up- or down-regulation of transcripts anddisease-specific diagnosis. The RNA targeting effector proteins can beused for visualization of RNA in (living) cells using e.g. fluorescentmicroscopy or flow cytometry, such as fluorescence-activated cellsorting (FACS) which allows for high-throughput screening of cells andrecovery of living cells following cell sorting. Further, expressionlevels of different transcripts can be assessed simultaneously understress, e.g. inhibition of cancer growth using molecular inhibitors orhypoxic conditions on cells. Another application would be to tracklocalization of transcripts to synaptic connections during a neuralstimulus using two photon microscopy.

In certain embodiments, the components or complexes according to theinvention as described herein can be used in multiplexed error-robustfluorescence in situ hybridization (MERFISH; Chen et al. Science; 2015;348(6233)), such as for instance with (fluorescently) labeled Cas13beffectors.

In Vitro Apex Labeling

Cellular processes depend on a network of molecular interactions amongprotein, RNA, and DNA. Accurate detection of protein-DNA and protein-RNAinteractions is key to understanding such processes. In vitro proximitylabeling technology employs an affinity tag combined with e.g. aphotoactivatable probe to label polypeptides and RNAs in the vicinity ofa protein or RNA of interest in vitro. After UV irradiation thephotoactivatable group reacts with proteins and other molecules that arein close proximity to the tagged molecule, thereby labelling them.Labelled interacting molecules can subsequently be recovered andidentified. The RNA targeting effector protein of the invention can forinstance be used to target a probe to a selected RNA sequence.

These applications could also be applied in animal models for in vivoimaging of disease relevant applications or difficult-to culture celltypes.

Use of RNA-Targeting Effector Protein in RNA Origami/In Vitro AssemblyLines—Combinatorics

RNA origami refers to nanoscale folded structures for creatingtwo-dimensional or three-dimensional structures using RNA as integratedtemplate. The folded structure is encoded in the RNA and the shape ofthe resulting RNA is thus determined by the synthesized RNA sequence(Geary, et al. 2014. Science, 345 (6198). pp. 799-804). The RNA origamimay act as scaffold for arranging other components, such as proteins,into complexes. The RNA targeting effector protein of the invention canfor instance be used to target proteins of interest to the RNA origamiusing a suitable guide RNA.

These applications could also be applied in animal models for in vivoimaging of disease relevant applications or difficult-to culture celltypes.

Use of RNA-Targeting Effector Protein in RNA Isolation or Purification,Enrichment or Depletion

It is further envisages that the RNA targeting effector protein whencomplexed to RNA can be used to isolate and/or purify the RNA. The RNAtargeting effector protein can for instance be fused to an affinity tagthat can be used to isolate and/or purify the RNA-RNA targeting effectorprotein complex. Such applications are for instance useful in theanalysis of gene expression profiles in cells.

In particular embodiments, it can be envisaged that the RNA targetingeffector proteins can be used to target a specific noncoding RNA (ncRNA)thereby blocking its activity, providing a useful functional probe. Incertain embodiments, the effector protein as described herein may beused to specifically enrich for a particular RNA (including but notlimited to increasing stability, etc.), or alternatively to specificallydeplete a particular RNA (such as without limitation for instanceparticular splice variants, isoforms, etc.).Interrogation of lincRNA Function and Other Nuclear RNAs

Current RNA knockdown strategies such as siRNA have the disadvantagethat they are mostly limited to targeting cytosolic transcripts sincethe protein machinery is cytosolic. The advantage of a RNA targetingeffector protein of the present invention, an exogenous system that isnot essential to cell function, is that it can be used in anycompartment in the cell. By fusing a NLS signal to the RNA targetingeffector protein, it can be guided to the nucleus, allowing nuclear RNAsto be targeted. It is for instance envisaged to probe the function oflincRNAs. Long intergenic non-coding RNAs (lincRNAs) are a vastlyunderexplored area of research. Most lincRNAs have as of yet unknownfunctions which could be studies using the RNA targeting effectorprotein of the invention.

Identification of RNA Binding Proteins

Identifying proteins bound to specific RNAs can be useful forunderstanding the roles of many RNAs. For instance, many lincRNAsassociate with transcriptional and epigenetic regulators to controltranscription. Understanding what proteins bind to a given lincRNA canhelp elucidate the components in a given regulatory pathway. A RNAtargeting effector protein of the invention can be designed to recruit abiotin ligase to a specific transcript in order to label locally boundproteins with biotin. The proteins can then be pulled down and analyzedby mass spectrometry to identify them.

Assembly of Complexes on RNA and Substrate Shuttling

RNA targeting effector proteins of the invention can further be used toassemble complexes on RNA. This can be achieved by functionalizing theRNA targeting effector protein with multiple related proteins (e.g.components of a particular synthesis pathway). Alternatively, multipleRNA targeting effector proteins can be functionalized with suchdifferent related proteins and targeted to the same or adjacent targetRNA. Useful application of assembling complexes on RNA are for instancefacilitating substrate shuttling between proteins.

Synthetic Biology

The development of biological systems have a wide utility, including inclinical applications. It is envisaged that the programmable RNAtargeting effector proteins of the invention can be used fused to splitproteins of toxic domains for targeted cell death, for instance usingcancer-linked RNA as target transcript. Further, pathways involvingprotein-protein interaction can be influenced in synthetic biologicalsystems with e.g. fusion complexes with the appropriate effectors suchas kinases or other enzymes.

Protein Splicing: Inteins

Protein splicing is a post-translational process in which an interveningpolypeptide, referred to as an intein, catalyzes its own excision fromthe polypeptides Hacking it, referred to as exteins, as well assubsequent ligation of the exteins. The assembly of two or more RNAtargeting effector proteins as described herein on a target transcriptcould be used to direct the release of a split intein (Topilina andMills Mob DNA. 2014 Feb. 4; 5(1):5), thereby allowing for directcomputation of the existence of a mRNA transcript and subsequent releaseof a protein product, such as a metabolic enzyme or a transcriptionfactor (for downstream actuation of transcription pathways). Thisapplication may have significant relevance in synthetic biology (seeabove) or large-scale bioproduction (only produce product under certainconditions).

Inducible, Dosed and Self-Inactivating Systems

In one embodiment, fusion complexes comprising an RNA targeting effectorprotein of the invention and an effector component are designed to beinducible, for instance light inducible or chemically inducible. Suchinducibility allows for activation of the effector component at adesired moment in time.

Light inducibility is for instance achieved by designing a fusioncomplex wherein CRY2 PHR/CIBN pairing is used for fusion. This system isparticularly useful for light induction of protein interactions inliving cells (Konermann S, et al. Nature. 2013; 500:472-476).

Chemical inducibility is for instance provided for by designing a fusioncomplex wherein FKBP/FRB (FK506 binding protein/FKBP rapamycin binding)pairing is used for fusion. Using this system rapamycin is required forbinding of proteins (Zetsche et al. Nat Biotechnol. 2015; 33(2):139-42describes the use of this system for Cas9).

Further, when introduced in the cell as DNA, the RNA targeting effectorprotein of the inventions can be modulated by inducible promoters, suchas tetracycline or doxycycline controlled transcriptional activation(Tet-On and Tet-Off expression system), hormone inducible geneexpression system such as for instance an ecdysone inducible geneexpression system and an arabinose-inducible gene expression system.When delivered as RNA, expression of the RNA targeting effector proteincan be modulated via a riboswitch, which can sense a small molecule liketetracycline (as described in Goldfless et al. Nucleic Acids Res. 2012;40(9):e64).

In one embodiment, the delivery of the RNA targeting effector protein ofthe invention can be modulated to change the amount of protein or crRNAin the cell, thereby changing the magnitude of the desired effect or anyundesired off-target effects.

In one embodiment, the RNA targeting effector proteins described hereincan be designed to be self-inactivating. When delivered to a cell asRNA, either mRNA or as a replication RNA therapeutic (Wrobleska et alNat Biotechnol. 2015 August; 33(8): 839-841), they can self-inactivateexpression and subsequent effects by destroying the own RNA, therebyreducing residency and potential undesirable effects.

For further in vivo applications of RNA targeting effector proteins asdescribed herein, reference is made to Mackay J P et al (Nat Struct MolBiol. 2011 Mar.; 18(3):256-61), Nelles et al (Bioessays. 2015 July;37(7):732-9) and Abil Z and Zhao H (Mol Biosyst. 2015 Oct.;11(10):2658-65), which are incorporated herein by reference. Inparticular, the following applications are envisaged in certainembodiments of the invention, preferably in certain embodiments by usingcatalytically inactive Cas13b: enhancing translation (e.g.Cas13b—translation promotion factor fusions (e.g. eIF4 fusions));repressing translation (e.g. gRNA targeting ribosome binding sites);exon skipping (e.g. gRNAs targeting splice donor and/or acceptor sites);exon inclusion (e.g. gRNA targeting a particular exon splice donorand/or acceptor site to be included or Cas13b fused to or recruitingspliceosome components (e.g. U1 snRNA)); accessing RNA localization(e.g. Cas13b—marker fusions (e.g. EGFP fusions)); altering RNAlocalization (e.g. Cas13b—localization signal fusions (e.g. NLS or NESfusions)); RNA degradation (in this case no catalytically inactiveCas13b is to be used if relied on the activity of Cas13b, alternativelyand for increased specificity, a split Cas13b may be used); inhibitionof non-coding RNA function (e.g. miRNA), such as by degradation orbinding of gRNA to functional sites (possibly titrating out at specificsites by relocalization by Cas13b-signal sequence fusions).

As described herein before and demonstrated in the Examples, Cas13bfunction is robust to 5′ or 3′ extensions of the crRNA and to extensionof the crRNA loop. It is therefore envisages that MS2 loops and otherrecruitment domains can be added to the crRNA without affecting complexformation and binding to target transcripts. Such modifications to thecrRNA for recruitment of various effector domains are applicable in theuses of a RNA targeted effector proteins described above.

Cas13b is capable of mediating resistance to RNA phages. It is thereforeenvisaged that Cas13b can be used to immunize, e.g. animals, humans andplants, against RNA-only pathogens, including but not limited to Ebolavirus and Zika virus.

In certain embodiments, Cas13b can process (cleave) its own array. Thisapplies to both the wildtype Cas13b protein and the mutated Cas13bprotein containing one or more mutated amino acid residues asherein-discussed. It is therefore envisaged that multiple crRNAsdesigned for different target transcripts and/or applications can bedelivered as a single pre-crRNA or as a single transcript driven by onepromotor. Such method of delivery has the advantages that it issubstantially more compact, easier to synthesize and easier to deliveryin viral systems. It will be understood that exact amino acid positionsmay vary for orthologues of a herein Cas13b can be adequately determinedby protein alignment, as is known in the art, and as described hereinelsewhere. Aspects of the invention also encompass methods and uses ofthe compositions and systems described herein in genome engineering,e.g. for altering or manipulating the expression of one or more genes orthe one or more gene products, in prokaryotic or eukaryotic cells, invitro, in vivo or ex vivo.

In an aspect, the invention provides methods and compositions formodulating, e.g., reducing, expression of a target RNA in cells. In thesubject methods, a Cas13b system of the invention is provided thatinterferes with transcription, stability, and/or translation of an RNA.

In certain embodiments, an effective amount of Cas13b system is used tocleave RNA or otherwise inhibit RNA expression. In this regard, thesystem has uses similar to siRNA and shRNA, thus can also be substitutedfor such methods. The method includes, without limitation, use of aCas13b system as a substitute for e.g., an interfering ribonucleic acid(such as an siRNA or shRNA) or a transcription template thereof, e.g., aDNA encoding an shRNA. The Cas13b system is introduced into a targetcell, e.g., by being administered to a mammal that includes the targetcell.

Advantageously, a Cas13b system of the invention is specific. Forexample, whereas interfering ribonucleic acid (such as an siRNA orshRNA) polynucleotide systems are plagued by design and stability issuesand off-target binding, a Cas13b system of the invention can be designedwith high specificity.

Destabilized Cas13b

In certain embodiments, the effector protein according to the inventionas described herein is associated with or fused to a destabilizationdomain (DD). In some embodiments, the DD is ER50. A correspondingstabilizing ligand for this DD is, in some embodiments, 4HT. As such, insome embodiments, one of the at least one DDs is ER50 and a stabilizingligand therefor is 4HT or CMP8. In some embodiments, the DD is DHFR50. Acorresponding stabilizing ligand for this DD is, in some embodiments,TMP. As such, in some embodiments, one of the at least one DDs is DHFR50and a stabilizing ligand therefor is TMP. In some embodiments, the DD isER50. A corresponding stabilizing ligand for this DD is, in someembodiments, CMP8. CMP8 may therefore be an alternative stabilizingligand to 4HT in the ER50 system. While it may be possible that CMP8 and4HT can/should be used in a competitive matter, some cell types may bemore susceptible to one or the other of these two ligands, and from thisdisclosure and the knowledge in the art the skilled person can use CMP8and/or 4HT.

In some embodiments, one or two DDs may be fused to the N-terminal endof the Cas13b with one or two DDs fused to the C-terminal of the Cas13b.In some embodiments, the at least two DDs are associated with the Cas13band the DDs are the same DD, i.e. the DDs are homologous. Thus, both (ortwo or more) of the DDs could be ER50 DDs. This is preferred in someembodiments. Alternatively, both (or two or more) of the DDs could beDHFR50 DDs. This is also preferred in some embodiments. In someembodiments, the at least two DDs are associated with the Cas13b and theDDs are different DDs, i.e. the DDs are heterologous. Thus, one of theDDS could be ER50 while one or more of the DDs or any other DDs could beDHFR50. Having two or more DDs which are heterologous may beadvantageous as it would provide a greater level of degradation control.A tandem fusion of more than one DD at the N or C-term may enhancedegradation; and such a tandem fusion can be, for exampleER50-ER50-Cas13b or DHFR-DHFR-Cas13b It is envisaged that high levels ofdegradation would occur in the absence of either stabilizing ligand,intermediate levels of degradation would occur in the absence of onestabilizing ligand and the presence of the other (or another)stabilizing ligand, while low levels of degradation would occur in thepresence of both (or two of more) of the stabilizing ligands. Controlmay also be imparted by having an N-terminal ER50 DD and a C-terminalDHFR50 DD.

In some embodiments, the fusion of the Cas13b with the DD comprises alinker between the DD and the Cas13b. In some embodiments, the linker isa GlySer linker. In some embodiments, the DD-Cas13b further comprises atleast one Nuclear Export Signal (NES). In some embodiments, theDD-Cas13b comprises two or more NESs. In some embodiments, the DD-Cas13bcomprises at least one Nuclear Localization Signal (NLS). This may be inaddition to an NES. In some embodiments, the Cas13b comprises orconsists essentially of or consists of a localization (nuclear import orexport) signal as, or as part of, the linker between the Cas13b and theDD. HA or Flag tags are also within the ambit of the invention aslinkers. Applicants use NLS and/or NES as linker and also use GlycineSerine linkers as short as GS up to (GGGGS)3.

Destabilizing domains have general utility to confer instability to awide range of proteins; see, e.g., Miyazaki, J Am Chem Soc. Mar. 7,2012; 134(9): 3942-3945, incorporated herein by reference. CMP8 or4-hydroxytamoxifen can be destabilizing domains. More generally, Atemperature-sensitive mutant of mammalian DHFR (DHFRts), a destabilizingresidue by the N-end rule, was found to be stable at a permissivetemperature but unstable at 37° C. The addition of methotrexate, ahigh-affinity ligand for mammalian DHFR, to cells expressing DHFRtsinhibited degradation of the protein partially. This was an importantdemonstration that a small molecule ligand can stabilize a proteinotherwise targeted for degradation in cells. A rapamycin derivative wasused to stabilize an unstable mutant of the FRB domain of mTOR (FRB*)and restore the function of the fused kinase, GSK-3β.6,7 This systemdemonstrated that ligand-dependent stability represented an attractivestrategy to regulate the function of a specific protein in a complexbiological environment. A system to control protein activity can involvethe DD becoming functional when the ubiquitin complementation occurs byrapamycin induced dimerization of FK506-binding protein and FKBP12.Mutants of human FKBP12 or ecDHFR protein can be engineered to bemetabolically unstable in the absence of their high-affinity ligands,Shield-1 or trimethoprim (TMP), respectively. These mutants are some ofthe possible destabilizing domains (DDs) useful in the practice of theinvention and instability of a DD as a fusion with a Cas13b confers tothe Cas13b degradation of the entire fusion protein by the proteasome.Shield-1 and TMP bind to and stabilize the DD in a dose-dependentmanner. The estrogen receptor ligand binding domain (ERLBD, residues305-549 of ERS1) can also be engineered as a destabilizing domain. Sincethe estrogen receptor signaling pathway is involved in a variety ofdiseases such as breast cancer, the pathway has been widely studied andnumerous agonist and antagonists of estrogen receptor have beendeveloped. Thus, compatible pairs of ERLBD and drugs are known. Thereare ligands that bind to mutant but not wild-type forms of the ERLBD. Byusing one of these mutant domains encoding three mutations (L384M,M421G, G521R)12, it is possible to regulate the stability of anERLBD-derived DD using a ligand that does not perturb endogenousestrogen-sensitive networks. An additional mutation (Y537S) can beintroduced to further destabilize the ERLBD and to configure it as apotential DD candidate. This tetra-mutant is an advantageous DDdevelopment. The mutant ERLBD can be fused to a Cas13b and its stabilitycan be regulated or perturbed using a ligand, whereby the Cas13b has aDD. Another DD can be a 12-kDa (107-amino-acid) tag based on a mutatedFKBP protein, stabilized by Shield1 ligand; see, e.g., Nature Methods 5,(2008). For instance a DD can be a modified FK506 binding protein 12(FKBP12) that binds to and is reversibly stabilized by a synthetic,biologically inert small molecule, Shield-1; see, e.g., Banaszynski L A,Chen L C, Maynard-Smith L A, Ooi A G, Wandless T J. A rapid, reversible,and tunable method to regulate protein function in living cells usingsynthetic small molecules. Cell. 2006; 126:995-1004; Banaszynski L A,Sellmyer M A, Contag C H, Wandless T J, Thorne S H. Chemical control ofprotein stability and function in living mice. Nat Med. 2008;14:1123-1127; Maynard-Smith L A, Chen L C, Banaszynski L A, Ooi A G,Wandless T J. A directed approach for engineering conditional proteinstability using biologically silent small molecules. The Journal ofbiological chemistry. 2007; 282:24866-24872; and Rodriguez, Chem Biol.Mar. 23, 2012; 19(3): 391-398-all of which are incorporated herein byreference and may be employed in the practice of the invention inselected a DD to associate with a Cas13b in the practice of thisinvention. As can be seen, the knowledge in the art includes a number ofDDs, and the DD can be associated with, e.g., fused to, advantageouslywith a linker, to a Cas13b, whereby the DD can be stabilized in thepresence of a ligand and when there is the absence thereof the DD canbecome destabilized, whereby the Cas13b is entirely destabilized, or theDD can be stabilized in the absence of a ligand and when the ligand ispresent the DD can become destabilized; the DD allows the Cas13b andhence the CRISPR-Cas13b complex or system to be regulated orcontrolled-turned on or off so to speak, to thereby provide means forregulation or control of the system, e.g., in an in vivo or in vitroenvironment. For instance, when a protein of interest is expressed as afusion with the DD tag, it is destabilized and rapidly degraded in thecell, e.g., by proteasomes. Thus, absence of stabilizing ligand leads toa D associated Cas being degraded. When a new DD is fused to a proteinof interest, its instability is conferred to the protein of interest,resulting in the rapid degradation of the entire fusion protein. Peakactivity for Cas is sometimes beneficial to reduce off-target effects.Thus, short bursts of high activity are preferred. The present inventionis able to provide such peaks. In some senses the system is inducible.In some other senses, the system repressed in the absence of stabilizingligand and de-repressed in the presence of stabilizing ligand.

Cas13 Mutations

In certain embodiments, the effector protein (CRISPR enzyme; Cas13;effector protein) according to the invention as described herein is acatalytically inactive or dead Cas13 effector protein (dCas13). In someembodiments, the dCas13 effector comprises mutations in the nucleasedomain. In some embodiments, the dCas13 effector protein has beentruncated. To reduce the size of a fusion protein of the Cas13 effectorand the one or more functional domains, the C-terminus of the Cas13effector can be truncated while still maintaining its RNA bindingfunction. For example, at least 20 amino acids, at least 50 amino acids,at least 80 amino acids, or at least 100 amino acids, or at least 150amino acids, or at least 200 amino acids, or at least 250 amino acids,or at least 300 amino acids, or at least 350 amino acids, or up to 120amino acids, or up to 140 amino acids, or up to 160 amino acids, or upto 180 amino acids, or up to 200 amino acids, or up to 250 amino acids,or up to 300 amino acids, or up to 350 amino acids, or up to 400 aminoacids, may be truncated at the C-terminus of the Cas13b effector.Specific examples of Cas13 truncations include C-terminal Δ984-1090,C-terminal Δ1026-1090, and C-terminal Δ1053-1090, C-terminal Δ934-1090,C-terminal Δ884-1090, C-terminal Δ834-1090, C-terminal Δ784-1090, andC-terminal Δ734-1090, wherein amino acid positions correspond to aminoacid positions of Prevotella sp. P5 125 Cas13b protein. See FIG. 28 .

Modulating Cas13 Effector Proteins

The invention provides accessory proteins that modulate CRISPR proteinfunction. In certain embodiments, the accessory protein modulatescatalytic activity of a CRISPR protein. In an embodiment of theinvention an accessory protein modulates targeted, or sequence specific,nuclease activity. In an embodiment of the invention, an accessoryprotein modulates collateral nuclease activity. In an embodiment of theinvention, an accessory protein modulates binding to a target nucleicacid.

According to the invention, the nuclease activity to be modulated can bedirected against nucleic acids comprising or consisting of RNA,including without limitation mRNA, miRNA, siRNA and nucleic acidscomprising cleavable RNA linkages along with nucleotide analogs. In anembodiment of the invention, the nuclease activity to be modulated canbe directed against nucleic acids comprising or consisting of DNA,including without limitation nucleic acids comprising cleavable DNAlinkages and nucleic acid analogs.

In an embodiment of the invention, an accessory protein enhances anactivity of a CRISPR protein. In certain such embodiments, the accessoryprotein comprises a HEPN domain and enhances RNA cleavage. In certainembodiments, the accessory protein inhibits an activity of a CRISPRprotein. In certain such embodiments, the accessory protein comprises aninactivated HEPN domain or lacks an HEPN domain altogether.

According to the invention, naturally occurring accessory proteins ofType VI CRISPR systems comprise small proteins encoded at or near aCRISPR locus that function to modify an activity of a CRISPR protein. Ingeneral a CRISPR locus can be identified as comprising a putative CRISPRarray and/or encoding a putative CRISPR effector protein. In anembodiment, an effector protein can be from 800 to 2000 amino acids, orfrom 900 to 1800 amino acids, or from 950 to 1300 amino acids. In anembodiment, an accessory protein can be encoded within 25 kb, or within20 kb or within 15 kb, or within 10 kb of a putative CRISPR effectorprotein or array, or from 2 kb to 10 kb from a putative CRISPR effectorprotein or array.

In an embodiment of the invention, an accessory protein is from 50 to300 amino acids, or from 100 to 300 amino acids or from 150 to 250 aminoacids or about 200 amino acids. Non-limiting examples of accessoryproteins include the csx27 and csx28 proteins identified herein.

Identification and use of a CRISPR accessory protein of the invention isindependent of CRISPR effector protein classification. Accessoryproteins of the invention can be found in association with or engineeredto function with a variety of CRISPR effector proteins. Examples ofaccessory proteins identified and used herein are representative ofCRISPR effector proteins generally. It is understood that CRISPReffector protein classification may involve homology, feature location(e.g., location of REC domains, NUC domains, HEPN sequences), nucleicacid target (e.g. DNA or RNA), absence or presence of tracr RNA,location of guide/spacer sequence 5′ or 3′ of a direct repeat, or othercriteria. In embodiments of the invention, accessory proteinidentification and use transcend such classifications.

In type VI CRISPR-Cas systems that target RNA, the Cas proteins usuallycomprise two conserved HEPN domains which are involved in RNA cleavage.In certain embodiments, the Cas protein processes crRNA to generatemature crRNA. The guide sequence of the crRNA recognizes target RNA witha complementary sequence and the Cas protein degrades the target strand.More particularly, in certain embodiments, upon target binding, the Casprotein undergoes a structural rearrangement that brings two HEPNdomains together to form an active HEPN catalytic site and the targetRNA is then cleaved. The location of the catalytic site near the surfaceof the Cas protein allows non-specific collateral ssRNA cleavage.

In certain embodiments, accessory proteins are instrumental inincreasing or reducing target and/or collateral RNA cleavage. Withoutbeing bound by theory, an accessory protein that activates CRISPRactivity (e.g., a csx28 protein or ortholog or variant comprising a HEPNdomain) can be envisioned as capable of interacting with a Cas proteinand combining its HEPN domain with a HEPN domain of the Cas protein toform an active HEPN catalytic site, whereas an inhibitory accessoryprotein (e.g. csx27 with lacks an HEPN domain) can be envisioned ascapable of interacting with a Cas protein and reducing or blocking aconformation of the Cas protein that would bring together two HEPNdomains.

According to the invention, in certain embodiments, enhancing activityof a Type VI Cas protein or complex thereof comprises contacting theType VI Cas protein or complex thereof with an accessory protein fromthe same organism that activates the Cas protein. In other embodiments,enhancing activity of a Type VI Cas protein of complex thereof comprisescontacting the Type VI Cas protein or complex thereof with an activatoraccessory protein from a different organism within the same subclass(e.g., Type VI-b). In other embodiments, enhancing activity of a Type VICas protein or complex thereof comprises contacting the Type VI Casprotein or complex thereof with an accessory protein not within thesubclass (e.g., a Type VI Cas protein other than Type VI-b with a TypeVI-b accessory protein or vice-versa).

According to the invention, in certain embodiments, repressing activityof a Type VI Cas protein or complex thereof comprises contacting theType VI Cas protein or complex thereof with an accessory protein fromthe same organism that represses the Cas protein. In other embodiments,repressing activity of a Type VI Cas protein or complex thereofcomprises contacting the Type VI Cas protein or complex thereof with anrepressor accessory protein from a different organism within the samesubclass (e.g., Type VI-b). In other embodiments, repressing activity ofa Type VI Cas protein or complex thereof comprises contacting the TypeVI Cas protein or complex thereof with an repressor accessory proteinnot within the subclass (e.g., a Type VI Cas protein other than TypeVI-b with a Type VI-b repressor accessory protein or vice-versa).

In certain embodiments where the Type VI Cas protein and the Type VIaccessory protein are from the same organism, the two proteins willfunction together in an engineered CRISPR system. In certainembodiments, it will be desirable to alter the function of theengineered CRISPR system, for example by modifying either or both of theproteins or their expression. In embodiments where the Type VI Casprotein and the Type VI accessory protein are from different organismswhich may be within the same class or different classes, the proteinsmay function together in an engineered CRISPR system but it will oftenbe desired or necessary to modify either or both of the proteins tofunction together.

Accordingly, in certain embodiments of the invention either or both of aCas protein and an accessory protein may be modified to adjust aspectsof protein-protein interactions between the Cas protein and accessoryprotein. In certain embodiments, either or both of a Cas protein and anaccessory protein may be modified to adjust aspects of protein-nucleicacid interactions. Ways to adjust protein-protein interactions andprotein-nucleic acid interaction include without limitation, fittingmolecular surfaces, polar interactions, hydrogen bonds, and modulatingvan der Waals interactions. In certain embodiments, adjustingprotein-protein interactions or protein-nucleic acid binding comprisesincreasing or decreasing binding interactions. In certain embodiments,adjusting protein-protein interactions or protein-nucleic acid bindingcomprises modifications that favor or disfavor a conformation of theprotein or nucleic acid.

By “fitting”, is meant determining including by automatic, orsemi-automatic means, interactions between one or more atoms of a Cas13protein and at least one atoms of a Cas13 accessory protein, or betweenone or more atoms of a Cas13 protein and one or more atoms of a nucleicacid, or between one or more atoms of a Cas13 accessory protein and anucleic acid, and calculating the extent to which such interactions arestable. Interactions include attraction and repulsion, brought about bycharge, steric considerations and the like.

The three-dimensional structure of Type VI CRISPR protein or complexthereof or a Type VI CRISPR accessory protein or complex thereofprovides in the context of the instant invention an additional tool foridentifying additional mutations in orthologs of Cas13. The crystalstructure can also be basis for the design of new and specific Cas13sand Cas13 accessory proteins. Various computer-based methods for fittingare described further. Binding interactions of Cas13s, accessoryproteins, and nucleic acids can be examined through the use of computermodeling using a docking program. Docking programs are known; forexample GRAM, DOCK or AUTODOCK (see Walters et al. Drug Discovery Today,vol. 3, no. 4 (1998), 160-178, and Dunbrack et al. Folding and Design 2(1997), 27-42). This procedure can include computer fitting to ascertainhow well the shape and the chemical structure of the binding partners.Computer-assisted, manual examination of the active site or binding siteof a Type VI system may be performed. Programs such as GRID (P.Goodford, J. Med. Chem, 1985, 28, 849-57)—a program that determinesprobable interaction sites between molecules with various functionalgroups—may also be used to analyze the active site or binding site topredict partial structures of binding compounds. Computer programs canbe employed to estimate the attraction, repulsion or steric hindrance ofthe two binding partners, e.g., components of a Type VI CRISPR system,or a nucleic acid molecule and a component of a Type VI CRISPR system.

Amino acid substitutions may be made on the basis of differences orsimilarities in amino acid properties (such as polarity, charge,solubility, hydrophobicity, hydrophilicity, and/or the amphipathicnature of the residues) and it is therefore useful to group amino acidstogether in functional groups. Amino acids may be grouped together basedon the properties of their side chains alone. In comparing orthologs,ther are likely to be residues conserved for structural or catalyticreasons. These sets may be described in the form of a Venn diagram(Livingstone C. D. and Barton G. J. (1993) “Protein sequence alignments:a strategy for the hierarchical analysis of residue conservation”Comput. Appl. Biosci. 9: 745-756) (Taylor W. R. (1986) “Theclassification of amino acid conservation” J. Theor. Biol. 119;205-218). Conservative substitutions may be made, for example accordingto the table below which describes a generally accepted Venn diagramgrouping of amino acids.

Set Sub-set Hydro- F W Y H K M I L Aromatic F W Y H phobic V A G C(SEQ ID NO: 20) (SEQ ID NO: 17) Aliphatic I L V Polar W Y H K R E D CCharged H K R E D S T N Q (SEQ ID NO: 21) (SEQ ID NO: 18) PositivelyH K R charged Negatively E D charged Small V C A G S P T N Tiny A G S D(SEQ ID NO: 19)

In an engineered Cas13 system, modification may comprise modification ofone or more amino acid residues of the Cas13 protein and/or may comprisemodification of one or more amino acid residues of the Cas13 accessoryprotein.

In an engineered Cas13 system, modification may comprise modification ofone or more amino acid residues located in a region which comprisesresidues which are positively charged in the unmodified Cas13 proteinand/or Cas13 accessory protein.

In an engineered Cas13 system, modification may comprise modification ofone or more amino acid residues which are positively charged in theunmodified Cas13 protein and/or Cas13 accessory protein.

In an engineered Cas13 system, modification may comprise modification ofone or more amino acid residues which are not positively charged in theunmodified Cas13 protein and/or Cas13 accessory protein.

The modification may comprise modification of one or more amino acidresidues which are uncharged in the unmodified Cas13 protein and/orCas13 accessory protein.

The modification may comprise modification of one or more amino acidresidues which are negatively charged in the unmodified Cas13 proteinand/or Cas13 accessory protein.

The modification may comprise modification of one or more amino acidresidues which are hydrophobic in the unmodified Cas13 protein and/orCas13 accessory protein.

The modification may comprise modification of one or more amino acidresidues which are polar in the unmodified Cas13 protein and/or Cas13accessory protein.

The modification may comprise substitution of a hydrophobic amino acidor polar amino acid with a charged amino acid, which can be a negativelycharged or positively charged amino acid. The modification may comprisesubstitution of a negatively charged amino acid with a positivelycharged or polar or hydrophobic amino acid. The modification maycomprise substitution of a positively charged amino acid with anegatively charged or polar or hydrophobic amino acid.

Embodiments of the invention include sequences (both polynucleotide orpolypeptide) which may comprise homologous substitution (substitutionand replacement are both used herein to mean the interchange of anexisting amino acid residue or nucleotide, with an alternative residueor nucleotide) that may occur i.e., like-for-like substitution in thecase of amino acids such as basic for basic, acidic for acidic, polarfor polar, etc. Non-homologous substitution may also occur i.e., fromone class of residue to another or alternatively involving the inclusionof unnatural amino acids such as ornithine (hereinafter referred to asZ), diaminobutyric acid ornithine (hereinafter referred to as B),norleucine ornithine (hereinafter referred to as O), pyridylalanine,thienylalanine, naphthylalanine and phenylglycine. Variant amino acidsequences may include suitable spacer groups that may be insertedbetween any two amino acid residues of the sequence including alkylgroups such as methyl, ethyl or propyl groups in addition to amino acidspacers such as glycine or β-alanine residues. A further form ofvariation, which involves the presence of one or more amino acidresidues in peptoid form, may be well understood by those skilled in theart. For the avoidance of doubt, “the peptoid form” is used to refer tovariant amino acid residues wherein the α-carbon substituent group is onthe residue's nitrogen atom rather than the α-carbon. Processes forpreparing peptides in the peptoid form are known in the art, for exampleSimon R J et al., PNAS (1992) 89(20), 9367-9371 and Horwell D C, TrendsBiotechnol. (1995) 13(4), 132-134.

Homology modelling: Corresponding residues in other Cas13 orthologs canbe identified by the methods of Zhang et al., 2012 (Nature; 490(7421):556-60) and Chen et al., 2015 (PLoS Comput Biol; 11(5): e1004248)—acomputational protein-protein interaction (PPI) method to predictinteractions mediated by domain-motif interfaces. PrePPI (PredictingPPI), a structure based PPI prediction method, combines structuralevidence with non-structural evidence using a Bayesian statisticalframework. The method involves taking a pair of query proteins and usingstructural alignment to identify structural representatives thatcorrespond to either their experimentally determined structures orhomology models. Structural alignment is further used to identify bothclose and remote structural neighbours by considering global and localgeometric relationships. Whenever two neighbors of the structuralrepresentatives form a complex reported in the Protein Data Bank, thisdefines a template for modelling the interaction between the two queryproteins. Models of a complex are created by superimposing therepresentative structures on their corresponding structural neighbour inthe template. This approach is in Dey et al., 2013 (Prot Sci; 22:359-66).

Application of RNA Targeting-CRISPR System to Plants and YeastDefinitions:

In general, the term “plant” relates to any various photosynthetic,eukaryotic, unicellular or multicellular organism of the kingdom Plantaecharacteristically growing by cell division, containing chloroplasts,and having cell walls comprised of cellulose. The term plant encompassesmonocotyledonous and dicotyledonous plants. Specifically, the plants areintended to comprise without limitation angiosperm and gymnosperm plantssuch as acacia, alfalfa, amaranth, apple, apricot, artichoke, ash tree,asparagus, avocado, banana, barley, beans, beet, birch, beech,blackberry, blueberry, broccoli, Brussel's sprouts, cabbage, canola,cantaloupe, carrot, cassava, cauliflower, cedar, a cereal, celery,chestnut, cherry, Chinese cabbage, citrus, clementine, clover, coffee,corn, cotton, cowpea, cucumber, cypress, eggplant, elm, endive,eucalyptus, fennel, figs, fir, geranium, grape, grapefruit, groundnuts,ground cherry, gum hemlock, hickory, kale, kiwifruit, kohlrabi, larch,lettuce, leek, lemon, lime, locust, pine, maidenhair, maize, mango,maple, melon, millet, mushroom, mustard, nuts, oak, oats, oil palm,okra, onion, orange, an ornamental plant or flower or tree, papaya,palm, parsley, parsnip, pea, peach, peanut, pear, peat, pepper,persimmon, pigeon pea, pine, pineapple, plantain, plum, pomegranate,potato, pumpkin, radicchio, radish, rapeseed, raspberry, rice, rye,sorghum, safflower, sallow, soybean, spinach, spruce, squash,strawberry, sugar beet, sugarcane, sunflower, sweet potato, sweet corn,tangerine, tea, tobacco, tomato, trees, triticale, turf grasses,turnips, vine, walnut, watercress, watermelon, wheat, yams, yew, andzucchini. The term plant also encompasses Algae, which are mainlyphotoautotrophs unified primarily by their lack of roots, leaves andother organs that characterize higher plants.

The methods for modulating gene expression using the RNA targetingsystem as described herein can be used to confer desired traits onessentially any plant, A wide variety of plants and plant cell systemsmay be engineered for the desired physiological and agronomiccharacteristics described herein using the nucleic acid constructs ofthe present disclosure and the various transformation methods mentionedabove. In preferred embodiments, target plants and plant cells forengineering include, but are not limited to, those monocotyledonous anddicotyledonous plants, such as crops including grain crops (e.g., wheat,maize, rice, millet, barley), fruit crops (e.g., tomato, apple, pear,strawberry, orange), forage crops (e.g., alfalfa), root vegetable crops(e.g., carrot, potato, sugar beets, yam), leafy vegetable crops (e.g.,lettuce, spinach); flowering plants (e.g., petunia, rose,chrysanthemum), conifers and pine trees (e.g., pine fir, spruce); plantsused in phytoremediation (e.g., heavy metal accumulating plants); oilcrops (e.g., sunflower, rape seed) and plants used for experimentalpurposes (e.g., Arabidopsis). Thus, the methods and CRISPR-Cas systemscan be used over a broad range of plants, such as for example withdicotyledonous plants belonging to the orders Magnoliales, Illiciales,Laurales, Piperales, Aristolochiaceae, Nymphaeales, Ranunculales,Papaveraceae, Sarraceniaceae, Trochodendrales, Hamamelidales, Eucommiae,Leitneriales, Myricales, Fagales, Casuarinaceae, Caryophyllales,Batales, Polygonales, Plumbaginales, Dilleniales, Theales, Malvales,Urticales, Lecythidales, Violates, Salicales, Capparales, Ericales,Diapensales, Ebenales, Primulales, Rosales, Fabales, Podostemales,Haloragales, Myrtales, Cornales, Proteales, San tales, Rafflesiales,Celastrales, Euphorbiales, Rhamnales, Sapindales, Juglandales,Geraniales, Polygalales, Umbellales, Gentianales, Polemoniales,Lamiales, Plantaginales, Scrophulariales, Campanulales, Rubiales,Dipsacales, and Asterales; the methods and CRISPR-Cas systems can beused with monocotyledonous plants such as those belonging to the ordersAlismatales, Hydrocharitales, Najadales, Triuridales, Commelinales,Eriocaulales, Restionales, Poales, Juncales, Cyperales, Typhales,Bromeliales, Zingiberales, Arecales, Cyclanthales, Pandanales, Arales,Liliales, and Orchid ales, or with plants belonging to Gymnospermae, e.gthose belonging to the orders Pinales, Ginkgoales, Cycadales,Araucariales, Cupressales and Gnetales.

The RNA targeting CRISPR systems and methods of use described herein canbe used over a broad range of plant species, included in thenon-(imitative list of dicot, monocot or gymnosperm genera hereunder:Atropa, Alseodaphne, Anaccrrcliurn, Arachis, Beilschmiedia, Brassica,Carthamus, Cocculus, Croton, Cucumis, Citrus, Citrullus, Capsicum,Catharanthus, Cocos, Coffea, Cucurbita, Daucus, Duguetia, Eschscholzia,Ficus, Fragaria, Glaucium, Glycine, Gossypium, Helianthus, Hevea,Hyoscyamus, Lactuca, Landolphia, Linum, Liam Lycopersicon, Lupinus,Manihot, Majorana, Malus, Medicago, Nicotiana, Olea, Parthenium,Papaver, Persea, Phaseolus, Pisiacia, Pisum, Pyrus, Prunus, Raphanus,Ricinus, Senecio, Sinomenium, Stephania, Sinapis, Solanum, Theobroma,Trifolium, Trigonella, Vicia, Vinca, Vilis, and Vigna; and the generaAllium, Andropogon, Eragrostis, Asparagus, Avena, Cynodon, Elaeis,Festuca, Festulolium, Hemerocallis, Hordeum, Lemna, Lolium, Musa, Oryza,Panicum, Pennisetum, Phleum, Poa, Secale, Sorghum, Triticum, Zea, Abies,Cunninghamia, Ephedra, Picea, Pinus, and Pseudotsuga.

The RNA targeting CRISPR systems and methods of use can also be usedover a broad range of “algae” or “algae cells”; including for examplealgea selected from several eukaryotic phyla, including the Rhodophyta(red algae), Chlorophyta (green algae), Phacophyta (brown algae),Bacillariophyta (diatoms), Eustigmatophyta and dinoflagellates as wellas the prokaryotic phylum Cyanobacteria (blue-green algae). The term“algae” includes for example algae selected from: Amphora, Anabaena,Ankistrodesmus, Botryococcus, Chaetoceros, Chlamydomonas, Chlorella,Chlorococcum, Cyclotella, Cylindrotheca, Dunaliella, Emiliana, Euglena,Haematococcus, Isochrysis, Monochrysis, Monoraphidium, Nannochloris,Nannochioropsis, Navicula, Nephrochloris, Neplaroselmis, Nitzschia,Nodularia, Nostoc, Ochromonas, Oocystis, Oscillatoria, Pavlova,Phaeodactylum, Platymonas, Pleurochrysis, Porphyra Pseudoanabaena,Pyramimonas, Stichococcus, Synechococcus, Synechocystis, Tetraselmis,Thalassiosira, and Trichodesmium.

A part of a plant, i.e., a “plant tissue” may be treated according tothe methods of the present invention to produce an improved plant. Planttissue also encompasses plant cells. The term “plant cell” as usedherein refers to individual units of a living plant, either in an intactwhole plant or in an isolated form grown in in vitro tissue cultures, onmedia or agar, in suspension in a growth media or buffer or as a part ofhigher organized unites, such as, for example, plant tissue, a plantorgan, or a whole plant.

A “protoplast” refers to a plant cell that has had its protective cellwall completely or partially removed using, for example, mechanical orenzymatic means resulting in an intact biochemical competent unit ofliving plant that can reform their cell wall, proliferate and regenerategrow into a whole plant under proper growing conditions.

The term “transformation” broadly refers to the process by which a planthost is genetically modified by the introduction of DNA by means ofAgrobacteria or one of a variety of chemical or physical methods. Asused herein, the term “plant host” refers to plants, including anycells, tissues, organs, or progeny of the plants. Many suitable planttissues or plant cells can be transformed and include, but are notlimited to, protoplasts, somatic embryos, pollen, leaves, seedlings,stems, calli, stolons, microtubers, and shoots. A plant tissue alsorefers to any clone of such a plant, seed, progeny, propagule whethergenerated sexually or asexually, and descendents of any of these, suchas cuttings or seed.

The term “transformed” as used herein, refers to a cell, tissue, organ,or organism into which a foreign DNA molecule, such as a construct, hasbeen introduced. The introduced DNA molecule may be integrated into thegenomic DNA of the recipient cell, tissue, organ, or organism such thatthe introduced DNA molecule is transmitted to the subsequent progeny. Inthese embodiments, the “transformed” or “transgenic” cell or plant mayalso include progeny of the cell or plant and progeny produced from abreeding program employing such a transformed plant as a parent in across and exhibiting an altered phenotype resulting from the presence ofthe introduced DNA molecule. Preferably, the transgenic plant is fertileand capable of transmitting the introduced DNA to progeny through sexualreproduction.

The term “progeny”, such as the progeny of a transgenic plant, is onethat is born of, begotten by, or derived from a plant or the transgenicplant. The introduced DNA molecule may also be transiently introducedinto the recipient cell such that the introduced DNA molecule is notinherited by subsequent progeny and thus not considered “transgenic”.Accordingly, as used herein, a “non-transgenic” plant or plant cell is aplant which does not contain a foreign DNA stably integrated into itsgenome.

The term “plant promoter” as used herein is a promoter capable ofinitiating transcription in plant cells, whether or not its origin is aplant cell. Exemplary suitable plant promoters include, but are notlimited to, those that are obtained from plants, plant viruses, andbacteria such as Agrobacterium or Rhizobium which comprise genesexpressed in plant cells.

As used herein, a “fungal cell” refers to any type of eukaryotic cellwithin the kingdom of fungi. Phyla within the kingdom of fungi includeAscomycota, Basidiomycota, Blastocladiomycota, Chytridiomycota,Glomeromycota, Microsporidia, and Neocallimastigomycota. Fungal cellsmay include yeasts, molds, and filamentous fungi. In some embodiments,the fungal cell is a yeast cell.

As used herein, the term “yeast cell” refers to any fungal cell withinthe phyla Ascomycota and Basidiomycota. Yeast cells may include buddingyeast cells, fission yeast cells, and mold cells. Without being limitedto these organisms, many types of yeast used in laboratory andindustrial settings are part of the phylum Ascomycota. In someembodiments, the yeast cell is an S. cerervisiae, Kluyveromycesmarxianus, or Issatchenkia orientalis cell. Other yeast cells mayinclude without limitation Candida spp. (e.g., Candida albicans),Yarrowia spp. (e.g., Yarrowia lipolytica), Pichia spp. (e.g., Pichiapastoris), Kluyveromyces spp. (e.g., Kluyveromyces lactis andKluyveromyces marxianus), Neurospora spp. (e.g., Neurospora crassa),Fusarium spp. (e.g., Fusarium oxysporum), and Issatchenkia spp. (e.g.,Issatchenkia orientalis, a.k.a. Pichia kudriavzevii and Candidaacidothermophilum). In some embodiments, the fungal cell is afilamentous fungal cell. As used herein, the term “filamentous fungalcell” refers to any type of fungal cell that grows in filaments, i.e.,hyphae or mycelia. Examples of filamentous fungal cells may includewithout limitation Aspergillus spp. (e.g., Aspergillus niger),Trichoderma spp. (e.g., Trichoderma reesei), Rhizopus spp. (e.g.,Rhizopus oryzae), and Mortierella spp. (e.g., Mortierella isabellina).

In some embodiments, the fungal cell is an industrial strain. As usedherein, “industrial strain” refers to any strain of fungal cell used inor isolated from an industrial process, e.g., production of a product ona commercial or industrial scale. Industrial strain may refer to afungal species that is typically used in an industrial process, or itmay refer to an isolate of a fungal species that may be also used fornon-industrial purposes (e.g., laboratory research). Examples ofindustrial processes may include fermentation (e.g., in production offood or beverage products), distillation, biofuel production, productionof a compound, and production of a polypeptide. Examples of industrialstrains may include, without limitation, JAY270 and ATCC4124.

In some embodiments, the fungal cell is a polyploid cell. As usedherein, a “polyploid” cell may refer to any cell whose genome is presentin more than one copy. A polyploid cell may refer to a type of cell thatis naturally found in a polyploid state, or it may refer to a cell thathas been induced to exist in a polyploid state (e.g., through specificregulation, alteration, inactivation, activation, or modification ofmeiosis, cytokinesis, or DNA replication). A polyploid cell may refer toa cell whose entire genome is polyploid, or it may refer to a cell thatis polyploid in a particular genomic locus of interest. Without wishingto be bound to theory, it is thought that the abundance of guide-RNA maymore often be a rate-limiting component in genome engineering ofpolyploid cells than in haploid cells, and thus the methods using theCas13b CRISPR system described herein may take advantage of using acertain fungal cell type.

In some embodiments, the fungal cell is a diploid cell. As used herein,a “diploid” cell may refer to any cell whose genome is present in twocopies. A diploid cell may refer to a type of cell that is naturallyfound in a diploid state, or it may refer to a cell that has beeninduced to exist in a diploid state (e.g., through specific regulation,alteration, inactivation, activation, or modification of meiosis,cytokinesis, or DNA replication). For example, the S. cerevisiae strainS228C may be maintained in a haploid or diploid state. A diploid cellmay refer to a cell whose entire genome is diploid, or it may refer to acell that is diploid in a particular genomic locus of interest. In someembodiments, the fungal cell is a haploid cell. As used herein, a“haploid” cell may refer to any cell whose genome is present in onecopy. A haploid cell may refer to a type of cell that is naturally foundin a haploid state, or it may refer to a cell that has been induced toexist in a haploid state (e.g., through specific regulation, alteration,inactivation, activation, or modification of meiosis, cytokinesis, orDNA replication). For example, the S. cerevisiae strain S228C may bemaintained in a haploid or diploid state. A haploid cell may refer to acell whose entire genome is haploid, or it may refer to a cell that ishaploid in a particular genomic locus of interest.

As used herein, a “yeast expression vector” refers to a nucleic acidthat contains one or more sequences encoding an RNA and/or polypeptideand may further contain any desired elements that control the expressionof the nucleic acid(s), as well as any elements that enable thereplication and maintenance of the expression vector inside the yeastcell. Many suitable yeast expression vectors and features thereof areknown in the art; for example, various vectors and techniques areillustrated in in Yeast Protocols, 2nd edition, Xiao, W., ed. (HumanaPress, New York, 2007) and Buckholz, R. G. and Gleeson, M. A. (1991)Biotechnology (NY) 9(11): 1067-72. Yeast vectors may contain, withoutlimitation, a centromeric (CEN) sequence, an autonomous replicationsequence (ARS), a promoter, such as an RNA Polymerase III promoter,operably linked to a sequence or gene of interest, a terminator such asan RNA polymerase III terminator, an origin of replication, and a markergene (e.g., auxotrophic, antibiotic, or other selectable markers).Examples of expression vectors for use in yeast may include plasmids,yeast artificial chromosomes, 2μ plasmids, yeast integrative plasmids,yeast replicative plasmids, shuttle vectors, and episomal plasmids.

Stable Integration of RNA Targeting CRISP System Components in theGenome of Plants and Plant Cells

In particular embodiments, it is envisaged that the polynucleotidesencoding the components of the RNA targeting CRISPR system areintroduced for stable integration into the genome of a plant cell. Inthese embodiments, the design of the transformation vector or theexpression system can be adjusted depending on when, where and underwhat conditions the guide RNA and/or the RNA targeting gene(s) areexpressed.

In particular embodiments, it is envisaged to introduce the componentsof the RNA targeting CRISPR system stably into the genomic DNA of aplant cell. Additionally or alternatively, it is envisaged to introducethe components of the RNA targeting CRISPR system for stable integrationinto the DNA of a plant organelle such as, but not limited to a plastid,e mitochondrion or a chloroplast.

The expression system for stable integration into the genome of a plantcell may contain one or more of the following elements: a promoterelement that can be used to express the guide RNA and/or RNA targetingenzyme in a plant cell; a 5′ untranslated region to enhance expression;an intron element to further enhance expression in certain cells, suchas monocot cells; a multiple-cloning site to provide convenientrestriction sites for inserting the one or more guide RNAs and/or theRNA targeting gene sequences and other desired elements; and a 3′untranslated region to provide for efficient termination of theexpressed transcript.

The elements of the expression system may be on one or more expressionconstructs which are either circular such as a plasmid or transformationvector, or non-circular such as linear double stranded DNA.

In a particular embodiment, a RNA targeting CRISPR expression systemcomprises at least:

-   -   (a) a nucleotide sequence encoding a guide RNA (gRNA) that        hybridizes with a target sequence in a plant, and wherein the        guide RNA comprises a guide sequence and a direct repeat        sequence, and    -   (b) a nucleotide sequence encoding a RNA targeting protein,    -   wherein components (a) or (b) are located on the same or on        different constructs, and whereby the different nucleotide        sequences can be under control of the same or a different        regulatory element operable in a plant cell.

DNA construct(s) containing the components of the RNA targeting CRISPRsystem, may be introduced into the genome of a plant, plant part, orplant cell by a variety of conventional techniques. The processgenerally comprises the steps of selecting a suitable host cell or hosttissue, introducing the construct(s) into the host cell or host tissue,and regenerating plant cells or plants therefrom.

In particular embodiments, the DNA construct may be introduced into theplant cell using techniques such as but not limited to electroporation,microinjection, aerosol beam injection of plant cell protoplasts, or theDNA constructs can be introduced directly to plant tissue usingbiolistic methods, such as DNA particle bombardment (see also Fu et al.,Transgenic Res. 2000 Feb.; 9(1):11-9). The basis of particle bombardmentis the acceleration of particles coated with gene/s of interest towardcells, resulting in the penetration of the protoplasm by the particlesand typically stable integration into the genome. (see e.g. Klein et al,Nature (1987), Klein et al, Bio/Technology (1992), Casas et al, Proc.Natl. Acad. Sci. USA (1993).).

In particular embodiments, the DNA constructs containing components ofthe RNA targeting CRISPR system may be introduced into the plant byAgrobacterium-mediated transformation. The DNA constructs may becombined with suitable T-DNA flanking regions and introduced into aconventional Agrobacterium tumefaciens host vector. The foreign DNA canbe incorporated into the genome of plants by infecting the plants or byincubating plant protoplasts with Agrobacterium bacteria, containing oneor more Ti (tumor-inducing) plasmids. (see e.g. Fraley et al., (1985),Rogers et al., (1987) and U.S. Pat. No. 5,563,055).

Plant Promoters

In order to ensure appropriate expression in a plant cell, thecomponents of the Cas13b CRISPR system described herein are typicallyplaced under control of a plant promoter, i.e. a promoter operable inplant cells. The use of different types of promoters is envisaged.

A constitutive plant promoter is a promoter that is able to express theopen reading frame (ORF) that it controls in all or nearly all of theplant tissues during all or nearly all developmental stages of the plant(referred to as “constitutive expression”). One non-limiting example ofa constitutive promoter is the cauliflower mosaic virus 35S promoter.The present invention envisages methods for modifying RNA sequences andas such also envisages regulating expression of plant biomolecules. Inparticular embodiments of the present invention it is thus advantageousto place one or more elements of the RNA targeting CRISPR system underthe control of a promoter that can be regulated. “Regulated promoter”refers to promoters that direct gene expression not constitutively, butin a temporally- and/or spatially-regulated manner, and includestissue-specific, tissue-preferred and inducible promoters. Differentpromoters may direct the expression of a gene in different tissues orcell types, or at different stages of development, or in response todifferent environmental conditions. In particular embodiments, one ormore of the RNA targeting CRISPR components are expressed under thecontrol of a constitutive promoter, such as the cauliflower mosaic virus35S promoter issue-preferred promoters can be utilized to targetenhanced expression in certain cell types within a particular planttissue, for instance vascular cells in leaves or roots or in specificcells of the seed. Examples of particular promoters for use in the RNAtargeting CRISPR system-are found in Kawamata et al., (1997) Plant CellPhysiol 38:792-803; Yamamoto et al., (1997) Plant J 12:255-65; Hire etal, (1992) Plant Mol Biol 20:207-18, Kuster et al, (1995) Plant Mol Biol29:759-72, and Capana et al., (1994) Plant Mo Biol 25:681-91.

Examples of promoters that are inducible and that allow forspatiotemporal control of gene editing or gene expression may use a formof energy. The form of energy may include but is not limited to soundenergy, electromagnetic radiation, chemical energy and/or thermalenergy. Examples of inducible systems include tetracycline induciblepromoters (Tet-On or Tet-Off), small molecule two-hybrid transcriptionactivations systems (FKBP, ABA, etc), or light inducible systems(Phytochrome, LOV domains, or cryptochrome), such as a Light InducibleTranscriptional Effector (LITE) that direct changes in transcriptionalactivity in a sequence-specific manner. The components of a lightinducible system may include a RNA targeting Cas13b, a light-responsivecytochrome heterodimer (e.g. from Arabidopsis thaliana), and atranscriptional activation/repression domain. Further examples ofinducible DNA binding proteins and methods for their use are provided inU.S. 61/736,465 and U.S. 61/721,283, which is hereby incorporated byreference in its entirety.

In particular embodiments, transient or inducible expression can beachieved by using, for example, chemical-regulated promotors, i.e.whereby the application of an exogenous chemical induces geneexpression. Modulating of gene expression can also be Obtained by achemical-repressible promoter, where application of the chemicalrepresses gene expression. Chemical inducible promoters include, but arenot limited to, the maize ln2-2 promoter, activated by benzenesulfonamide herbicide safeners (De Vevlder et al., (1997) Plant CellPhysiol 38:568-77), the maize GST promoter (GST-11-27, WO93/01294),activated by hydrophobic electrophilic compounds used as pre-emergentherbicides, and the tobacco PR-1 a promoter (Ono et al., (2004) BiosciBiotechnol Biocheni 68:803-7) activated by salicylic acid. Promoterswhich are regulated by antibiotics, such as tetracycline-inducible andtetracycline-repressible promoters (Gatz et al., (1991) Mol Gen Genet227:229-37; U.S. Pat. Nos. 5,814,618 and 5,789,156) can also be usedherein.

Translocation to and/or Expression in Specific Plant Organelles

The expression system may comprise elements for translocation to and/orexpression in a specific plant organelle.

Chloroplast Targeting

In particular embodiments, it is envisaged that the RNA targeting CRISPRsystem is used to specifically modify expression and/or translation ofchloroplast genes or to ensure expression in the chloroplast. For thispurpose use is made of chloroplast transformation methods orcompartmentalization of the RNA targeting CRISPR components to thechloroplast. For instance, the introduction of genetic modifications inthe plastid genome can reduce biosafety issues such as gene flow throughpollen.

Methods of chloroplast transformation are known in the art and includeParticle bombardment, PEG treatment, and microinjection. Additionally,methods involving the translocation of transformation cassettes from thenuclear genome to the plastid can be used as described in WO2010/061186.

Alternatively, it is envisaged to target one or more of the RNAtargeting CRISPR components to the plant chloroplast. This is achievedby incorporating in the expression construct a sequence encoding achloroplast transit peptide (CTP) or plastid transit peptide, operablylinked to the 5′ region of the sequence encoding the RNA targetingprotein. The CTP is removed in a processing step during translocationinto the chloroplast. Chloroplast targeting of expressed proteins iswell known to the skilled artisan (see for instance Protein Transportinto Chloroplasts, 2010, Annual Review of Plant Biology, Vol. 61:157-180). In such embodiments it is also desired to target the one ormore guide RNAs to the plant chloroplast. Methods and constructs whichcan be used for translocating guide RNA into the chloroplast by means ofa chloroplast localization sequence are described, for instance, in US20040142476, incorporated herein by reference. Such variations ofconstructs can be incorporated into the expression systems of theinvention to efficiently translocate the RNA targeting-guide RNA(s).

Introduction of Polynucleotides Encoding the CRISPR-RNA Targeting Systemin Algal Cells.

Transgenic algae (or other plants such as rape) may be particularlyuseful in the production of vegetable oils or biofuels such as alcohols(especially methanol and ethanol) or other products. These may beengineered to express or overexpress high levels of oil or alcohols foruse in the oil or biofuel industries.

U.S. Pat. No. 8,945,839 describes a method for engineering Micro-Algae(Chlamydomonas reinhardtii cells) species) using Cas9. Using similartools, the methods of the RNA targeting CRISPR system described hereincan be applied on Chlamydomonas species and other algae. In particularembodiments, RNA targeting protein and guide RNA(s) are introduced inalgae expressed using a vector that expresses RNA targeting proteinunder the control of a constitutive promoter such as Hsp70A-Rbc S2 orBeta2-tubulin. Guide RNA is optionally delivered using a vectorcontaining T7 promoter. Alternatively, RNA targeting mRNA and in vitrotranscribed guide RNA can be delivered to algal cells. Electroporationprotocols are available to the skilled person such as the standardrecommended protocol from the GeneArt Chlamydomonas Engineering kit.

Introduction of Polynucleotides Encoding RNA Targeting Components inYeast Cells

In particular embodiments, the invention relates to the use of the RNAtargeting CRISPR system for RNA editing in yeast cells. Methods fortransforming yeast cells which can be used to introduce polynucleotidesencoding the RNA targeting CRISPR system components are well known tothe artisan and are reviewed by Kawai et al., 2010, Bioeng Bugs. 2010November-December; 1(6): 395-403). Non-limiting examples includetransformation of yeast cells by lithium acetate treatment (which mayfurther include carrier DNA and PEG treatment), bombardment or byelectroporation.

Transient Expression of RNA Targeting CRISP System Components in Plantsand Plant Cell

In particular embodiments, it is envisaged that the guide RNA and/or RNAtargeting gene are transiently expressed in the plant cell. In theseembodiments, the RNA targeting CRISPR system can ensure modification ofRNA target molecules only when both the guide RNA and the RNA targetingprotein is present in a cell, such that gene expression can further becontrolled. As the expression of the RNA targeting enzyme is transient,plants regenerated from such plant cells typically contain no foreignDNA. In particular embodiments the RNA targeting enzyme is stablyexpressed by the plant cell and the guide sequence is transientlyexpressed.

In particularly preferred embodiments, the RNA targeting CRISPR systemcomponents can be introduced in the plant cells using a plant viralvector (Scholthof et al. 1996, Annu Rev Phytopathol. 1996; 34:299-323),In further particular embodiments, said viral vector is a vector from aDNA virus. For example, geminivirus (e.g., cabbage leaf curl virus, beanyellow dwarf virus, wheat dwarf virus, tomato leaf curl virus, maizestreak virus, tobacco leaf curl virus, or tomato golden mosaic virus) ornanovirus (e.g., Faba bean necrotic yellow virus). In other particularembodiments, said viral vector is a vector from an RNA virus. Forexample, tobravirus (e.g., tobacco rattle virus, tobacco mosaic virus),potexvirus (e.g., potato virus X), or hordeivirus (e.g., barley stripemosaic virus). The replicating genomes of plant viruses arenon-integrative vectors, which is of interest in the context of avoidingthe production of GMO plants.

In particular embodiments, the vector used for transient expression ofRNA targeting CRISPR constructs is for instance a pEAQ vector, which istailored for Agrobacterium-mediated transient expression (Sainsbury F.et al., Plant Biotechnol J. 2009 Sep.; 7(7):682-93) in the protoplast.Precise targeting of genomic locations was demonstrated using a modifiedCabbage Leaf Curl virus (CaLCuV) vector to express gRNAs in stabletransgenic plants expressing a Cas13b (see Scientific Reports 5, Articlenumber: 14926 (2015), doi:10.1038/srep14926).

In particular embodiments, double-stranded DNA fragments encoding theguide RNA or crRNA and/or the RNA targeting gene can be transientlyintroduced into the plant cell. In such embodiments, the introduceddouble-stranded DNA fragments are provided in sufficient quantity tomodify RNA molecule(s) in the cell but do not persist after acontemplated period of time has passed or after one or more celldivisions. Methods for direct DNA transfer in plants are known by theskilled artisan (see for instance Davey et al, Plant Mol Biol. 1989Sep.; 13(3):273-85.)

In other embodiments, an RNA polynucleotide encoding the RNA targetingprotein is introduced into the plant cell, which is then translated andprocessed by the host cell generating the protein in sufficient quantityto modify the RNA molecule(s) cell (in the presence of at least oneguide RNA) but which does not persist after a contemplated period oftime has passed or after one or more cell divisions. Methods forintroducing mRNA to plant protoplasts for transient expression are knownby the skilled artisan (see for instance in Gallie, Plant Cell Reports(1993), 13; 119-122). Combinations of the different methods describedabove are also envisaged.

Delivery of RNA Targeting CRISPR Components to the Plant Cell

In particular embodiments, it is of interest to deliver one or morecomponents of the RNA targeting CRISPR system directly to the plantcell. This is of interest, inter alia, for the generation ofnon-transgenic plants. In particular embodiments, one or more of the RNAtargeting components is prepared outside the plant or plant cell anddelivered to the cell. For instance in particular embodiments, the RNAtargeting protein is prepared in vitro prior to introduction to theplant cell. RNA targeting protein can be prepared by various methodsknown by one of skill in the art and include recombinant production.After expression, the RNA targeting protein is isolated, refolded ifneeded, purified and optionally treated to remove any purification tags,such as a His-tag. Once crude, partially purified, or more completelypurified RNA targeting protein is obtained, the protein may beintroduced to the plant cell.

In particular embodiments, the RNA targeting protein is mixed with guideRNA targeting the RNA of interest to form a pre-assembledribonucleoprotein.

The individual components or pre-assembled ribonucleoprotein can beintroduced into the plant cell via electroporation, by bombardment withRNA targeting-associated gene product coated particles, by chemicaltransfection or by some other means of transport across a cell membrane.For instance, transfection of a plant protoplast with a pre-assembledCRISPR ribonucleoprotein has been demonstrated to ensure targetedmodification of the plant genome (as described by Woo et al. NatureBiotechnology, 2015; DOI: 10.1038/nbt.3389). These methods can bemodified to achieve targeted modification of RNA molecules in theplants.

In particular embodiments, the RNA targeting CRISPR system componentsare introduced into the plant cells using nanoparticles. The components,either as protein or nucleic acid or in a combination thereof, can beuploaded onto or packaged in nanoparticles and applied to the plants(such as for instance described in WO 2008/042156 and US 20130185823).In particular, embodiments of the invention comprise nanoparticlesuploaded with or packed with DNA molecule(s) encoding the RNA targetingprotein, DNA molecules encoding the guide RNA and/or isolated guide RNAas described in WO 2015/089419.

Further means of introducing one or more components of the RNA targetingCRISPR system to the plant cell is by using cell penetrating peptides(CPP). Accordingly, in particular, embodiments the invention comprisescompositions comprising a cell penetrating peptide linked to an RNAtargeting protein. In particular embodiments of the present invention,an RNA targeting protein and/or guide RNA(s) is coupled to one or moreCPPs to effectively transport them inside plant protoplasts (Ramakrishna(2014, Genome Res. 2014 Jun.;24(6):1020-7 for Cas9 in human cells). Inother embodiments, the RNA targeting gene and/or guide RNA(s) areencoded by one or more circular or non-circular DNA molecule(s) whichare coupled to one or more CPPs for plant protoplast delivery. The plantprotoplasts are then regenerated to plant cells and further to plants.CPPs are generally described as short peptides of fewer than 35 aminoacids either derived from proteins or from chimeric sequences which arecapable of transporting biomolecules across cell membrane in a receptorindependent manner. CPP can be cationic peptides, peptides havinghydrophobic sequences, amphipathic peptides, peptides havingproline-rich and anti microbial sequence, and chimeric or bipartitepeptides (Pooga and Langel 2005). CPPs are able to penetrate biologicalmembranes and as such trigger the movement of various biomoleculesacross cell membranes into the cytoplasm and to improve theirintracellular routing, and hence facilitate interaction of thebiolomolecule with the target. Examples of CPP include amongst others:Tat, a nuclear transcriptional activator protein required for viralreplication by HIV type1, penetratin, Kaposi fibroblast growth factor(FGF) signal peptide sequence, integrin β3 signal peptide sequence;polyarginine peptide Args sequence, Guanine rich-molecular transporters,sweet arrow peptide, etc.

Target RNA Envisaged for Plant, Algae or Fungal Applications

The target RNA, i.e. the RNA of interest, is the RNA to be targeted bythe present invention leading to the recruitment to, and the binding ofthe RNA targeting protein at, the target site of interest on the targetRNA. The target RNA may be any suitable form of RNA. This may include,in some embodiments, mRNA. In other embodiments, the target RNA mayinclude transfer RNA (tRNA) or ribosomal RNA (rRNA). In otherembodiments the target RNA may include interfering RNA (RNAi), microRNA.(miRNA), rnicroswitches, microzymes, satellite RNAs and RNA viruses. Thetarget RNA may be located in the cytoplasm of the plant cell, or in thecell nucleus or in a plant cell organelle such as a mitochondrion,chloroplast or plastid.

In particular embodiments, the RNA targeting CRISPR system is used tocleave RNA or otherwise inhibit RNA expression.

Use of RNA Targeting CRISPR System for Modulating Plant Gene ExpressionVia RNA Modulation

The RNA targeting protein may also be used, together with a suitableguide RNA, to target gene expression, via control of RNA processing. Thecontrol of RNA processing may include RNA processing reactions such asRNA splicing, including alternative splicing; viral replication inparticular of plant viruses, including viroids in plants and tRNAbiosynthesis. The RNA targeting protein in combination with a suitableguide RNA may also be used to control RNA activation (RNAa). RNAa leadsto the promotion of gene expression, so control of gene expression maybe achieved that way through disruption or reduction of RNAa and thusless promotion of gene expression.

The RNA targeting effector protein of the invention can further be usedfor antiviral activity in plants, in particular against RNA viruses. Theeffector protein can be targeted to the viral RNA using a suitable guideRNA selective for a selected viral RNA sequence. In particular, theeffector protein may be an active nuclease that cleaves RNA, such assingle stranded RNA. provided is therefore the use of an RNA targetingeffector protein of the invention as an antiviral agent. Examples ofviruses that can be counteracted in this way include, but are notlimited to, Tobacco mosaic virus (TMV), Tomato spotted wilt virus(TSWV), Cucumber mosaic virus (CAW), Potato virus Y (PVY), Cauliflowermosaic virus (CaMV) (RT virus), Plum pox virus (PPV), Brome mosaic virus(BMV) and Potato virus X (PVX).

Examples of modulating RNA expression in plants, algae or fungi, as analternative of targeted gene modification are described herein further.

Of particular interest is the regulated control of gene expressionthrough regulated cleavage of mRNA, This can be achieved by placingelements of the RNA targeting under the control of regulated promotersas described herein.

Use of the RNA Targeting CRISPR System to Restore the Functionality oftRNA Molecules.

Pring et al describe RNA editing in plant mitochondria and chloroplaststhat alters mRNA sequences to code for different proteins than the DNA.(Plant Mol. Biol. (1993) 21(6): 1163-1170. doi:10.1007/BF00023611). Inparticular embodiments of the invention, the elements of the RNAtargeting CRISPR system specifically forgetting mitochondrial andchloroplast mRNA can be introduced in a plant or plant cell to expressdifferent proteins in such plant cell organelles mimicking the processesoccurring in vivo.

Use of the RNA Targeting CRISPR System as an Alternative to RNAInterference to Inhibit RNA Expression.

The RNA targeting CRISPR system has uses similar to RNA inhibition orRNA interference, thus can also be substituted for such methods. Inparticular embodiment, the methods of the present invention include theuse of the RNA targeting CRISPR as a substitute for e.g. an interferingribonucleic acid (such as an siRNA or shRNA or a dsRNA). Examples ofinhibition of RNA expression in plants, algae or fungi as an alternativeof targeted gene modification are described herein further.

Use of the RNA Targeting CRISPR System to Control RNA Interference.

Control over interfering RNA or miRNA may help reduce off-target effects(OTE) seen with those approaches by reducing the longevity of theinterfering RNA or miRNA in vivo or in vitro. In particular embodiments,the target RNA may include interfering RNA, i.e. RNA involved in an RNAinterference pathway, such as shRNA, siRNA and so forth. In otherembodiments, the target RNA may include microRNA (miRNA) or doublestranded RNA (dsRNA).

In other particular embodiments, if the RNA targeting protein andsuitable guide RNA(s) are selectively expressed (for example spatiallyor temporally under the control of a regulated promoter, for example atissue- or cell cycle-specific promoter and/or enhancer) this can beused to ‘protect’ the cells or systems (in vivo or in vitro) from RNAiin those cells. This may be useful in neighbouring tissues or cellswhere RNAi is not required or for the purposes of comparison of thecells or tissues where the effector protein and suitable guide are andare not expressed (i.e. where the RNAi is not controlled and where itis, respectively). The RNA targeting protein may be used to control orbind to molecules comprising or consisting of RNA, such as ribozymes,ribosomes or riboswitches. In embodiments of the invention, the guideRNA can recruit the RNA targeting protein to these molecules so that theRNA targeting protein is able to bind to them.

The RNA targeting CRISPR system of the invention can be applied in areasof in-planta RNAi technologies, without undue experimentation, from thisdisclosure, including insect pest management, plant disease managementand management of herbicide resistance, as well as in plant assay andfor other applications (see, for instance Kim et al., in PesticideBiochemistry and Physiology (Impact Factor: 2.01). January 2015; 120.DOI: 10.1016/j.pestbp.2015.01.002; Sharma et al. in Academic Journals(2015), Vol. 12(18) pp 2303-2312); Green J. M, inPest ManagementScience, Vol 70(9), pp 1351-1357), because the present applicationprovides the foundation for informed engineering of the system.

Use of RNA Targeting CRISPR System to Modify Riboswitches and ControlMetabolic Regulation in Plants, Algae and Fungi

Riboswitches (also known as aptozymes) are regulatory segments ofmessenger RNA that bind small molecules and in turn regulate geneexpression. This mechanism allows the cell to sense the intracellularconcentration of these small molecules. A particular riboswitchtypically regulates its adjacent gene by altering the transcription, thetranslation or the splicing of this gene. Thus, in particularembodiments of the present invention, control of riboswitch activity isenvisaged through the use of the RNA targeting protein in combinationwith a suitable guide RNA to target the riboswitch. This may be throughcleavage of, or binding to, the riboswitch. In particular embodiments,reduction of riboswitch activity is envisaged. Recently, a riboswitchthat binds thiamin pyrophosphate (TPP) was characterized and found toregulate thiamin biosynthesis in plants and algae. Furthermore itappears that this element is an essential regulator of primarymetabolism in plants (Bocobza and Aharoni, Plant J. 2014 August;79(4):693-703. doi: 10.1111/tpj.12540. Epub 2014 Jun. 17). TPPriboswitches are also found in certain fungi, such as in Neurosporacrassa, where it controls alternative splicing to conditionally producean Upstream Open Reading Frame (uORF), thereby affecting the expressionof downstream genes (Cheah M T et al., (2007)Nature 447 (7143): 497-500.doi:10.1038/nature05769) The RNA targeting CRISPR system describedherein may be used to manipulate the endogenous riboswitch activity inplants, algae or fungi and as such alter the expression of downstreamgenes controlled by it. In particular embodiments, the RNA targetingCRISP system may be used in assaying riboswitch function in vivo or invitro and in studying its relevance for the metabolic network. Inparticular embodiments the RNA targeting CRISPR system may potentiallybe used for engineering of riboswitches as metabolite sensors in plantsand platforms for gene control.

Use of RNA Targeting CRISPR System in RNAi Screens for Plants, Algae orFungi

Identifying gene products whose knockdown is associated with phenotypicchanges, biological pathways can be interrogated and the constituentparts identified, via RNAi screens. In particular embodiments of theinvention, control may also be exerted over or during these screens byuse of the Guide 29 or Guide 30 protein and suitable guide RNA describedherein to remove or reduce the activity of the RNAi in the screen andthus reinstate the activity of the (previously interfered with) geneproduct (by removing or reducing the interference/repression).

Use of RNA Targeting Proteins for Visualization of RNA Molecules In Vivoand In Vitro

In particular embodiments, the invention provides a nucleic acid bindingsystem. In situ hybridization of RNA with complementary probes is apowerful technique. Typically fluorescent DNA oligonucleotides are usedto detect nucleic acids by hybridization. Increased efficiency has beenattained by certain modifications, such as locked nucleic acids (LNAs),but there remains a need for efficient and versatile alternatives. Assuch, labelled elements of the RNA targeting system can be used as analternative for efficient and adaptable system for in situhybridization.

Further Applications of the RNA Targeting CRISPR System in Plants andYeasts Use of RNA Targeting CRISPR System in Biofuel Production

The term “biofuel” as used herein is an alternative fuel made from plantand plant-derived resources. Renewable biofuels can be extracted fromorganic matter whose energy has been obtained through a process ofcarbon fixation or are made through the use or conversion of biomass.This biomass can be used directly for biofuels or can be converted toconvenient energy containing substances by thermal conversion, chemicalconversion, and biochemical conversion. This biomass conversion canresult in fuel in solid, liquid, or gas form. There are two types ofbiofuels: bioethanol and biodiesel. Bioethanol is mainly produced by thesugar fermentation process of cellulose (starch), which is mostlyderived from maize and sugar cane. Biodiesel on the other hand is mainlyproduced from oil crops such as rapeseed, palm, and soybean. Biofuelsare used mainly for transportation.

Enhancing Plant Properties for Biofuel Production

In particular embodiments, the methods using the RNA targeting CRISPRsystem as described herein are used to alter the properties of the cellwall in order to facilitate access by key hydrolysing agents for a moreefficient release of sugars for fermentation. In particular embodiments,the biosynthesis of cellulose and/or lignin are modified. Cellulose isthe major component of the cell wall. The biosynthesis of cellulose andlignin are co-regulated. By reducing the proportion of lignin in a plantthe proportion of cellulose can be increased. In particular embodiments,the methods described herein are used to downregulate ligninbiosynthesis in the plant so as to increase fermentable carbohydrates.More particularly, the methods described herein are used to downregulateat least a first lignin biosynthesis gene selected from the groupconsisting of 4-coumarate 3-hydroxylase (C3H), phenylalanineammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), hydroxycinnamoyltransferase (HCT), caffeic acid O-methyltransferase (COMT), caffeoyl CoA3-O-methyltransferase (CCoAOMT), ferulate 5-hydroxylase (F5H), cinnamylalcohol dehydrogenase (CAD), cinnamoyl CoA-reductase (CCR),4-coumarate-CoA ligase (4CL), monolignol-lignin-specificglycosyltransferase, and aldehyde dehydrogenase (ALDH) as disclosed inWO 2008/064289 A2.

In particular embodiments, the methods described herein are used toproduce plant mass that produces lower levels of acetic acid duringfermentation (see also WO 2010/096488).

Modifying Yeast for Biofuel Production

In particular embodiments, the RNA targeting enzyme provided herein isused for bioethanol production by recombinant micro-organisms. Forinstance, RNA targeting enzymes can be used to engineer micro-organisms,such as yeast, to generate biofuel or biopolymers from fermentablesugars and optionally to be able to degrade plant-derived lignocellulosederived from agricultural waste as a source of fermentable sugars. Moreparticularly, the invention provides methods whereby the RNA targetingCRISPR complex is used to modify the expression of endogenous genesrequired for biofuel production and/or to modify endogenous genes whymay interfere with the biofuel synthesis. More particularly the methodsinvolve stimulating the expression in a micro-organism such as a yeastof one or more nucleotide sequence encoding enzymes involved in theconversion of pyruvate to ethanol or another product of interest. Inparticular embodiments the methods ensure the stimulation of expressionof one or more enzymes which allows the micro-organism to degradecellulose, such as a cellulase. In yet further embodiments, the RNAtargeting CRISPR complex is used to suppress endogenous metabolicpathways which compete with the biofuel production pathway.

Modifying Algae and Plants for Production of Vegetable Oils or Biofuels

Transgenic algae or other plants such as rape may be particularly usefulin the production of vegetable oils or biofuels such as alcohols(especially methanol and ethanol), for instance. These may be engineeredto express or overexpress high levels of oil or alcohols for use in theoil or biofuel industries.

U.S. Pat. No. 8,945,839 describes a method for engineering Micro-Algae(Chlamydomonas reinhardtii cells) species) using Cas9. Using similartools, the methods of the RNA targeting CRISPR system described hereincan be applied on Chlamydomonas species and other algae. In particularembodiments, the RNA targeting effector protein and guide RNA areintroduced in algae expressed using a vector that expresses the RNAtargeting effector protein under the control of a constitutive promotersuch as Hsp70A-Rbc S2 or Beta2-tubulin. Guide RNA will be deliveredusing a vector containing T7 promoter. Alternatively, in vitrotranscribed guide RNA can be delivered to algae cells. Electroporationprotocol follows standard recommended protocol from the GeneArtChlamydomonas Engineering kit.

Particular Applications of the RNA Targeting Enzymes in Plants

In particular embodiments, present invention can be used as a therapyfor virus removal in plant systems as it is able to cleave viral RNA.Previous studies in human systems have demonstrated the success ofutilizing CRISPR in targeting the single strand RNA virus, hepatitis C(A. Price, et al., Proc. Natl. Acad. Sci, 2015). These methods may alsobe adapted for using the RNA targeting CRISPR system in plants.

Improved Plants

The present invention also provides plants and yeast cells obtainableand obtained by the methods provided herein. The improved plantsobtained by the methods described herein may be useful in food or feedproduction through the modified expression of genes which, for instanceensure tolerance to plant pests, herbicides, drought, low or hightemperatures, excessive water, etc.

The improved plants obtained by the methods described herein, especiallycrops and algae may be useful in food or feed production throughexpression of, for instance, higher protein, carbohydrate, nutrient orvitamin levels than would normally be seen in the wildtype. In thisregard, improved plants, especially pulses and tubers are preferred.

Improved algae or other plants such as rape may be particularly usefulin the production of vegetable oils or biofuels such as alcohols(especially methanol and ethanol), for instance. These may be engineeredto express or overexpress high levels of oil or alcohols for use in theoil or biofuel industries.

The invention also provides for improved parts of a plant. Plant partsinclude, but are not limited to, leaves, sterns, roots, tubers, seeds,endosperm, ovule, and pollen. Plant parts as envisaged herein may beviable, nonviable, regeneratable, and/or non-regeneratable.

It is also encompassed herein to provide plant cells and plantsgenerated according to the methods of the invention. Gametes, seeds,embryos, either zygotic or somatic, progeny or hybrids of plantscomprising the genetic modification, which are produced by traditionalbreeding methods, are also included within the scope of the presentinvention. Such plants may contain a heterologous or foreign DNAsequence inserted at or instead of a target sequence. Alternatively,such plants may contain only an alteration (mutation, deletion,insertion, substitution) in one or more nucleotides. As such, suchplants will only be different from their progenitor plants by thepresence of the particular modification.

In an embodiment of the invention, a Cas13b system is used to engineerpathogen resistant plants, for example by creating resistance againstdiseases caused by bacteria, fungi or viruses. In certain embodiments,pathogen resistance can be accomplished by engineering crops to producea Cas13b system that will be ingested by an insect pest, leading tomortality. In an embodiment of the invention, a Cas13b system is used toengineer abiotic stress tolerance. In another embodiment, a Cas13bsystem is used to engineer drought stress tolerance or salt stresstolerance, or cold or heat stress tolerance. Younis et al. 2014, Int. J.Biol. Sci. 10; 1150 reviewed potential targets of plant breedingmethods, all of which are amenable to correction or improvement throughuse of a Cas13b system described herein. Some non-limiting target cropsinclude Arabidopsis Zea mays is thaliana, Oryza sativa L, Prunusdomestica L., Gossypium hirsutum, Nicotiana rustica, Zea mays, Medicagosativa, Nicotiana benthamiana and Arabidopsis thaliana.

In an embodiment of the invention, a Cas13b system is used formanagement of crop pests. For example, a Cas13b system operable in acrop pest can be expressed from a plant host or transferred directly tothe target, for example using a viral vector.

In an embodiment, the invention provides a method of efficientlyproducing homozygous organisms from a heterozygous non-human startingorganism. In an embodiment, the invention is used in plant breeding. Inanother embodiment, the invention is used in animal breeding. In suchembodiments, a homozygous organism such as a plant or animal is made bypreventing or suppressing recombination by interfering with at least onetarget gene involved in double strand breaks, chromosome pairing and/orstrand exchange.

Application of the CAS13B Proteins in Optimized Functional RNA TargetingSystems

In an aspect the invention provides a system for specific delivery offunctional components to the RNA environment. This can be ensured usingthe CRISPR systems comprising the RNA targeting effector proteins of thepresent invention which allow specific targeting of different componentsto RNA. More particularly such components include activators orrepressors, such as activators or repressors of RNA translation,degradation, etc. Applications of this system are described elsewhereherein.

According to one aspect the invention provides non-naturally occurringor engineered composition comprising a guide RNA comprising a guidesequence capable of hybridizing to a target sequence in a genomic locusof interest in a cell, wherein the guide RNA is modified by theinsertion of one or more distinct RNA sequence(s) that bind an adaptorprotein. In particular embodiments, the RNA sequences may bind to two ormore adaptor proteins (e.g. aptamers), and wherein each adaptor proteinis associated with one or more functional domains. The guide RNAs of theCas13b enzymes described herein are shown to be amenable to modificationof the guide sequence. In particular embodiments, the guide RNA ismodified by the insertion of distinct RNA sequence(s) 5′ of the directrepeat, within the direct repeat, or 3′ of the guide sequence. Whenthere is more than one functional domain, the functional domains can besame or different, e.g., two of the same or two different activators orrepressors. In an aspect the invention provides a herein-discussedcomposition, wherein the one or more functional domains are attached tothe RNA targeting enzyme so that upon binding to the target RNA thefunctional domain is in a spatial orientation allowing for thefunctional domain to function in its attributed function; In an aspectthe invention provides a herein-discussed composition, wherein thecomposition comprises a CRISPR-Cas complex having at least threefunctional domains, at least one of which is associated with the RNAtargeting enzyme and at least two of which are associated with the gRNA.

Accordingly, in an aspect the invention provides non-naturally occurringor engineered CRISPR-Cas13b complex composition comprising the guide RNAas herein-discussed and a Cas13b which is an RNA targeting enzyme,wherein optionally the RNA targeting enzyme comprises at least onemutation, such that the RNA targeting enzyme has no more than 5% of thenuclease activity of the enzyme not having the at least one mutation,and optionally one or more comprising at least one or more nuclearlocalization sequences. In particular embodiments, the guide RNA isadditionally or alternatively modified so as to still ensure binding ofthe RNA targeting enzyme but to prevent cleavage by the RNA targetingenzyme (as detailed elsewhere herein).

In particular embodiments, the RNA targeting enzyme is a Cas13b enzymewhich has a diminished nuclease activity of at least 97%, or 100% ascompared with the Cas13b enzyme not having the at least one mutation. Inan aspect the invention provides a herein-discussed composition, whereinthe Cas13b enzyme comprises two or more mutations as otherwiseherein-discussed.

In particular embodiments, an RNA targeting system is provided asdescribed herein above comprising two or more functional domains. Inparticular embodiments, the two or more functional domains areheterologous functional domain. In particular embodiments, the systemcomprises an adaptor protein which is a fusion protein comprising afunctional domain, the fusion protein optionally comprising a linkerbetween the adaptor protein and the functional domain. In particularembodiments, the linker includes a GlySer linker. Additionally oralternatively, one or more functional domains are attached to the RNAeffector protein by way of a linker, optionally a GlySer linker. Inparticular embodiments, the one or more functional domains are attachedto the RNA targeting enzyme through one or both of the HEPN domains.

In an aspect the invention provides a herein-discussed composition,wherein the one or more functional domains associated with the adaptorprotein or the RNA targeting enzume is a domain capable of activating orrepressing RNA translation. In an aspect the invention provides aherein-discussed composition, wherein at least one of the one or morefunctional domains associated with the adaptor protein have one or moreactivities comprising methylase activity, demethylase activity,transcription activation activity, transcription repression activity,transcription release factor activity, histone modification activity,DNA integration activity RNA cleavage activity, DNA cleavage activity ornucleic acid binding activity, or molecular switch activity or chemicalinducibility or light inducibility.

In an aspect the invention provides a herein-discussed compositioncomprising an aptamer sequence. In particular embodiments, the aptamersequence is two or more aptamer sequences specific to the same adaptorprotein. In an aspect the invention provides a herein-discussedcomposition, wherein the aptamer sequence is two or more aptamersequences specific to different adaptor protein. In an aspect theinvention provides a herein-discussed composition, wherein the adaptorprotein comprises MS2, PP7, Qβ, F2, GA, fr, JP501, M12, R17, BZ13, JP34,JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, ϕCb5,ϕCb8r, ϕCb12r, ϕCb23r, 7s, PRR1. Accordingly, in particular embodiments,the aptamer is selected from a binding protein specifically binding anyone of the adaptor proteins listed above. In an aspect the inventionprovides a herein-discussed composition, wherein the cell is aeukaryotic cell. In an aspect the invention provides a herein-discussedcomposition, wherein the eukaryotic cell is a mammalian cell, a plantcell or a yeast cell, whereby the mammalian cell is optionally a mousecell. In an aspect the invention provides a herein-discussedcomposition, wherein the mammalian cell is a human cell.

In an aspect the invention provides a herein above-discussed compositionwherein there is more than one guide RNA or gRNA or crRNA, and thesetarget different sequences whereby when the composition is employed,there is multiplexing. In an aspect the invention provides a compositionwherein there is more than one guide RNA or gRNA or crRNA modified bythe insertion of distinct RNA sequence(s) that bind to one or moreadaptor proteins.

In an aspect the invention provides a herein-discussed compositionwherein one or more adaptor proteins associated with one or morefunctional domains is present and bound to the distinct RNA sequence(s)inserted into the guide RNA(s).

In an aspect the invention provides a herein-discussed compositionwherein the guide RNA is modified to have at least one non-codingfunctional loop; e.g., wherein the at least one non-coding functionalloop is repressive; for instance, wherein at least one non-codingfunctional loop comprises Alu.

In an aspect the invention provides a method for modifying geneexpression comprising the administration to a host or expression in ahost in vivo of one or more of the compositions as herein-discussed.

In an aspect the invention provides a herein-discussed method comprisingthe delivery of the composition or nucleic acid molecule(s) codingtherefor, wherein said nucleic acid molecule(s) are operatively linkedto regulatory sequence(s) and expressed in vivo. In an aspect theinvention provides a herein-discussed method wherein the expression invivo is via a lentivirus, an adenovirus, or an AAV.

In an aspect the invention provides a mammalian cell line of cells asherein-discussed, wherein the cell line is, optionally, a human cellline or a mouse cell line. In an aspect the invention provides atransgenic mammalian model, optionally a mouse, wherein the model hasbeen transformed with a herein-discussed composition or is a progeny ofsaid transformant.

In an aspect the invention provides a nucleic acid molecule(s) encodingguide RNA or the RNA targeting CRISPR-Cas13b complex or the compositionas herein-discussed. In an aspect the invention provides a vectorcomprising: a nucleic acid molecule encoding a guide RNA (gRNA) or crRNAcomprising a guide sequence capable of hybridizing to an RNA targetsequence in a cell, wherein the direct repeat of the gRNA or crRNA ismodified by the insertion of distinct RNA sequence(s) that bind(s) totwo or more adaptor proteins, and wherein each adaptor protein isassociated with one or more functional domains; or, wherein the gRNA ismodified to have at least one non-coding functional loop. In an aspectthe invention provides vector(s) comprising nucleic acid molecule(s)encoding: non-naturally occurring or engineered CRISPR-Cas13b complexcomposition comprising the gRNA or crRNA herein-discussed, and an RNAtargeting enzyme, wherein optionally the RNA targeting enzyme comprisesat least one mutation, such that the RNA targeting enzyme has no morethan 5% of the nuclease activity of the RNA targeting enzyme not havingthe at least one mutation, and optionally one or more comprising atleast one or more nuclear localization sequences. In an aspect a vectorcan further comprise regulatory element(s) operable in a eukaryotic celloperably linked to the nucleic acid molecule encoding the guide RNA(gRNA) or crRNA and/or the nucleic acid molecule encoding the RNAtargeting enzyme and/or the optional nuclear localization sequence(s).

In one aspect, the invention provides a kit comprising one or more ofthe components described herein. In some embodiments, the kit comprisesa vector system as described herein and instructions for using the kit.

In an aspect the invention provides a method of screening for gain offunction (GOF) or loss of function (LOF) or for screening non-codingRNAs or potential regulatory regions (e.g. enhancers, repressors)comprising the cell line of as herein-discussed or cells of the modelherein-discussed containing or expressing the RNA targeting enzyme andintroducing a composition as herein-discussed into cells of the cellline or model, whereby the gRNA or crRNA includes either an activator ora repressor, and monitoring for GOF or LOF respectively as to thosecells as to which the introduced gRNA or crRNA includes an activator oras to those cells as to which the introduced gRNA or crRNA includes arepressor.

In an aspect the invention provides a library of non-naturally occurringor engineered compositions, each comprising a RNA targeting CRISPR guideRNA (gRNA) or crRNA comprising a guide sequence capable of hybridizingto a target RNA sequence of interest in a cell, an RNA targeting enzyme,wherein the RNA targeting enzyme comprises at least one mutation, suchthat the RNA targeting enzyme has no more than 5% of the nucleaseactivity of the RNA targeting enzyme not having the at least onemutation, wherein the gRNA or crRNA is modified by the insertion ofdistinct RNA sequence(s) that bind to one or more adaptor proteins, andwherein the adaptor protein is associated with one or more functionaldomains, wherein the composition comprises one or more or two or moreadaptor proteins, wherein the each protein is associated with one ormore functional domains, and wherein the gRNAs or crRNAs comprise agenome wide library comprising a plurality of RNA targeting guide RNAs(gRNAs) or crRNAs. In an aspect the invention provides a library asherein-discussed, wherein the RNA targeting RNA targeting enzyme has adiminished nuclease activity of at least 97%, or 100% as compare withthe RNA targeting enzyme not having the at least one mutation. In anaspect the invention provides a library as herein-discussed, wherein theadaptor protein is a fusion protein comprising the functional domain. Inan aspect the invention provides a library as herein discussed, whereinthe gRNA or crRNA is not modified by the insertion of distinct RNAsequence(s) that bind to the one or two or more adaptor proteins. In anaspect the invention provides a library as herein discussed, wherein theone or two or more functional domains are associated with the RNAtargeting enzyme. In an aspect the invention provides a library asherein discussed, wherein the cell population of cells is a populationof eukaryotic cells. In an aspect the invention provides a library asherein discussed, wherein the eukaryotic cell is a mammalian cell, aplant cell or a yeast cell. In an aspect the invention provides alibrary as herein discussed, wherein the mammalian cell is a human cell.In an aspect the invention provides a library as herein discussed,wherein the population of cells is a population of embryonic stem (ES)cells.

In an aspect the invention provides a library as herein discussed,wherein the targeting is of about 100 or more RNA sequences. In anaspect the invention provides a library as herein discussed, wherein thetargeting is of about 1000 or more RNA sequences. In an aspect theinvention provides a library as herein discussed, wherein the targetingis of about 20,000 or more sequences. In an aspect the inventionprovides a library as herein discussed, wherein the targeting is of theentire transcriptome. In an aspect the invention provides a library asherein discussed, wherein the targeting is of a panel of targetsequences focused on a relevant or desirable pathway. In an aspect theinvention provides a library as herein discussed, wherein the pathway isan immune pathway. In an aspect the invention provides a library asherein discussed, wherein the pathway is a cell division pathway.

In one aspect, the invention provides a method of generating a modeleukaryotic cell comprising a gene with modified expression. In someembodiments, a disease gene is any gene associated an increase in therisk of having or developing a disease. In some embodiments, the methodcomprises (a) introducing one or more vectors encoding the components ofthe system described herein above into a eukaryotic cell, and (b)allowing a CRISPR complex to bind to a target polynucleotide so as tomodify expression of a gene, thereby generating a model eukaryotic cellcomprising modified gene expression.

The structural information provided herein allows for interrogation ofguide RNA or crRNA interaction with the target RNA and the RNA targetingenzyme permitting engineering or alteration of guide RNA structure tooptimize functionality of the entire RNA targeting CRISPR-Cas13b system.For example, the guide RNA or crRNA may be extended, without collidingwith the RNA targeting protein by the insertion of adaptor proteins thatcan bind to RNA. These adaptor proteins can further recruit effectorproteins or fusions which comprise one or more functional domains.

An aspect of the invention is that the above elements are comprised in asingle composition or comprised in individual compositions. Thesecompositions may advantageously be applied to a host to elicit afunctional effect on the genomic level.

The skilled person will understand that modifications to the guide RNAor crRNA which allow for binding of the adapter+functional domain butnot proper positioning of the adapter+functional domain (e.g. due tosteric hindrance within the three dimensial structure of the CRISPRCas13b complex) are modifications which are not intended. The one ormore modified guide RNA or crRNA may be modified, by introduction of adistinct RNA sequence(s) 5′ of the direct repeat, within the directrepeat, or 3′ of the guide sequence.

The modified guide RNA or crRNA, the inactivated RNA targeting enzyme(with or without functional domains), and the binding protein with oneor more functional domains, may each individually be comprised in acomposition and administered to a host individually or collectively.Alternatively, these components may be provided in a single compositionfor administration to a host. Administration to a host may be performedvia viral vectors known to the skilled person or described herein fordelivery to a host (e.g. lentiviral vector, adenoviral vector, AAVvector). As explained herein, use of different selection markers (e.g.for lentiviral gRNA or crRNA selection) and concentration of gRNA orcrRNA (e.g. dependent on whether multiple gRNAs or crRNAs are used) maybe advantageous for eliciting an improved effect.

Using the provided compositions, the person skilled in the art canadvantageously and specifically target single or multiple loci with thesame or different functional domains to elicit one or more genomicevents. The compositions may be applied in a wide variety of methods forscreening in libraries in cells and functional modeling in vivo (e.g.gene activation of lincRNA and identification of function;gain-of-function modeling; loss-of-function modeling; the use thecompositions of the invention to establish cell lines and transgenicanimals for optimization and screening purposes).

The current invention comprehends the use of the compositions of thecurrent invention to establish and utilize conditional or inducibleCRISPR Cas13b RNA targeting events. (See, e.g., Platt et al., Cell(2014), http://dx.doi.org/10.1016/j.cell.2014.09.014, or PCT patentpublications cited herein, such as WO 2014/093622 (PCT/US2013/074667),which are not believed prior to the present invention or application).

Guide RNA According to the Invention Comprising a Dead Guide Sequence

In one aspect, the invention provides guide sequences which are modifiedin a manner which allows for formation of the CRISPR Cas13b complex andsuccessful binding to the target, while at the same time, not eitherallowing for or not allowing for successful nuclease activity (i.e.without nuclease activity/without indel activity). For matters ofexplanation such modified guide sequences are referred to as “deadguides” or “dead guide sequences”. These dead guides or dead guidesequences can be thought of as catalytically inactive orconformationally inactive with regard to nuclease activity. Indeed, deadguide sequences may not sufficiently engage in productive base pairingwith respect to the ability to promote catalytic activity or todistinguish on-target and off-target binding activity. Briefly, theassay involves synthesizing a CRISPR target RNA and guide RNAscomprising mismatches with the target RNA, combining these with the RNAtargeting enzyme and analyzing cleavage based on gels based on thepresence of bands generated by cleavage products, and quantifyingcleavage based upon relative band intensities.

Hence, in a related aspect, the invention provides a non-naturallyoccurring or engineered composition RNA targeting CRISPR-Cas systemcomprising a functional RNA targeting enzyme as described herein, andguide RNA (gRNA) or crRNA wherein the gRNA or crRNA comprises a deadguide sequence whereby the gRNA is capable of hybridizing to a targetsequence such that the RNA targeting CRISPR-Cas system is directed to agenomic locus of interest in a cell without detectable RNA cleavageactivity of a non-mutant RNA targeting enzyme of the system. It is to beunderstood that any of the gRNAs or crRNAs according to the invention asdescribed herein elsewhere may be used as dead gRNAs/crRNAs comprising adead guide sequence.

The ability of a dead guide sequence to direct sequence-specific bindingof a CRISPR complex to an RNA target sequence may be assessed by anysuitable assay. For example, the components of a CRISPR Cas13b systemsufficient to form a CRISPR Cas13b complex, including the dead guidesequence to be tested, may be provided to a host cell having thecorresponding target sequence, such as by transfection with vectorsencoding the components of the system, followed by an assessment ofpreferential cleavage within the target sequence.

As explained further herein, several structural parameters allow for aproper framework to arrive at such dead guides. Dead guide sequences canbe typically shorter than respective guide sequences which result inactive RNA cleavage. In particular embodiments, dead guides are 5%, 10%,20%, 30%, 40%, 50%, shorter than respective guides directed to the same.

As explained below and known in the art, one aspect of gRNA or crRNA-RNAtargeting specificity is the direct repeat sequence, which is to beappropriately linked to such guides. In particular, this implies thatthe direct repeat sequences are designed dependent on the origin of theRNA targeting enzyme. Structural data available for validated dead guidesequences may be used for designing Cas13b specific equivalents.Structural similarity between, e.g., the orthologous nuclease domainsHEPN of two or more Cas13b effector proteins may be used to transferdesign equivalent dead guides. Thus, the dead guide herein may beappropriately modified in length and sequence to reflect such Cas13bspecific equivalents, allowing for formation of the CRISPR Cas13bcomplex and successful binding to the target RNA, while at the sametime, not allowing for successful nuclease activity.

Dead guides allow one to use gRNA or crRNA as a means for genetargeting, without the consequence of nuclease activity, while at thesame time providing directed means for activation or repression. GuideRNA or crRNA comprising a dead guide may be modified to further includeelements in a manner which allow for activation or repression of geneactivity, in particular protein adaptors (e.g. aptamers) as describedherein elsewhere allowing for functional placement of gene effectors(e.g. activators or repressors of gene activity). One example is theincorporation of aptamers, as explained herein and in the state of theart. By engineering the gRNA or crRNA comprising a dead guide toincorporate protein-interacting aptamers (Konermann et al.,“Genome-scale transcription activation by an engineered CRISPR-Cas9complex,” doi:10.1038/nature14136, incorporated herein by reference),one may assemble multiple distinct effector domains. Such may be modeledafter natural processes.

General Information

In embodiments of the invention the terms guide sequence and guide RNAand crRNA are used interchangeably as in foregoing cited documents suchas WO 2014/093622 (PCT/US2013/074667). In general, a guide sequence isany polynucleotide sequence having sufficient complementarity with atarget polynucleotide sequence to hybridize with the target sequence anddirect sequence-specific binding of a CRISPR complex to the targetsequence. In some embodiments, the degree of complementarity between aguide sequence and its corresponding target sequence, when optimallyaligned using a suitable alignment algorithm, is about or more thanabout 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimalalignment may be determined with the use of any suitable algorithm foraligning sequences, non-limiting example of which include theSmith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithmsbased on the Burrows-Wheeler Transform (e.g., the Burrows WheelerAligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies;available at www.novocraft.com), ELAND (Illumina, San Diego, CA), SOAP(available at soap.genomics.org.cn), and Maq (available atmaq.sourceforge.net). In some embodiments, a guide sequence is about ormore than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotidesin length. In some embodiments, a guide sequence is less than about 75,50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides in length.Preferably the guide sequence is 10-30 nucleotides long, such as 30nucleotides long. The ability of a guide sequence to directsequence-specific binding of a CRISPR complex to a target sequence maybe assessed by any suitable assay. For example, the components of aCRISPR system sufficient to form a CRISPR complex, including the guidesequence to be tested, may be provided to a host cell having thecorresponding target sequence, such as by transfection with vectorsencoding the components of the CRISPR sequence, followed by anassessment of preferential cleavage within the target sequence, such asby Surveyor assay as described herein. Similarly, cleavage of a targetpolynucleotide sequence may be evaluated in a test tube by providing thetarget sequence, components of a CRISPR complex, including the guidesequence to be tested and a control guide sequence different from thetest guide sequence, and comparing binding or rate of cleavage at thetarget sequence between the test and control guide sequence reactions.Other assays are possible, and will occur to those skilled in the art. Aguide sequence may be selected to target any target sequence. In someembodiments, the target sequence is a sequence within a genome of acell. Exemplary target sequences include those that are unique in thetarget genome.

In general, and throughout this specification, the term “vector” refersto a nucleic acid molecule capable of transporting another nucleic acidto which it has been linked. Vectors include, but are not limited to,nucleic acid molecules that are single-stranded, double-stranded, orpartially double-stranded; nucleic acid molecules that comprise one ormore free ends, no free ends (e.g., circular); nucleic acid moleculesthat comprise DNA, RNA, or both; and other varieties of polynucleotidesknown in the art. One type of vector is a “plasmid,” which refers to acircular double stranded DNA loop into which additional DNA segments canbe inserted, such as by standard molecular cloning techniques. Anothertype of vector is a viral vector, wherein virally-derived DNA or RNAsequences are present in the vector for packaging into a virus (e.g.,retroviruses, replication defective retroviruses, adenoviruses,replication defective adenoviruses, and adeno-associated viruses). Viralvectors also include polynucleotides carried by a virus for transfectioninto a host cell. Certain vectors are capable of autonomous replicationin a host cell into which they are introduced (e.g., bacterial vectorshaving a bacterial origin of replication and episomal mammalianvectors). Other vectors (e.g., non-episomal mammalian vectors) areintegrated into the genome of a host cell upon introduction into thehost cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively-linked. Such vectors are referred toherein as “expression vectors.” Vectors for and that result inexpression in a eukaryotic cell can be referred to herein as “eukaryoticexpression vectors.” Common expression vectors of utility in recombinantDNA techniques are often in the form of plasmids.

Recombinant expression vectors can comprise a nucleic acid of theinvention in a form suitable for expression of the nucleic acid in ahost cell, which means that the recombinant expression vectors includeone or more regulatory elements, which may be selected on the basis ofthe host cells to be used for expression, that is operatively-linked tothe nucleic acid sequence to be expressed. Within a recombinantexpression vector, “operably linked” is intended to mean that thenucleotide sequence of interest is linked to the regulatory element(s)in a manner that allows for expression of the nucleotide sequence (e.g.,in an in vitro transcription/translation system or in a host cell whenthe vector is introduced into the host cell).

The term “regulatory element” is intended to include promoters,enhancers, internal ribosomal entry sites (IRES), and other expressioncontrol elements (e.g., transcription termination signals, such aspolyadenylation signals and poly-U sequences). Such regulatory elementsare described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY:METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).Regulatory elements include those that direct constitutive expression ofa nucleotide sequence in many types of host cell and those that directexpression of the nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). A tissue-specific promoter maydirect expression primarily in a desired tissue of interest, such asmuscle, neuron, bone, skin, blood, specific organs (e.g., liver,pancreas), or particular cell types (e.g., lymphocytes). Regulatoryelements may also direct expression in a temporal-dependent manner, suchas in a cell-cycle dependent or developmental stage-dependent manner,which may or may not also be tissue or cell-type specific. In someembodiments, a vector comprises one or more pol III promoter (e.g., 1,2, 3, 4, 5, or more pol III promoters), one or more pol II promoters(e.g., 1, 2, 3, 4, 5, or more pol II promoters), one or more pol Ipromoters (e.g., 1, 2, 3, 4, 5, or more pol I promoters), orcombinations thereof. Examples of pol III promoters include, but are notlimited to, U6 and H1 promoters. Examples of pol II promoters include,but are not limited to, the retroviral Rous sarcoma virus (RSV) LTRpromoter (optionally with the RSV enhancer), the cytomegalovirus (CMV)promoter (optionally with the CMV enhancer) [see, e.g., Boshart et al,Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductasepromoter, the β-actin promoter, the phosphoglycerol kinase (PGK)promoter, and the EF1α promoter. Also encompassed by the term“regulatory element” are enhancer elements, such as WPRE; CMV enhancers;the R-U5′ segment in LTR of HTLV-I (Mol. Cell. Biol., Vol. 8(1), p.466-472, 1988); SV40 enhancer; and the intron sequence between exons 2and 3 of rabbit β-globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), p.1527-31, 1981). It will be appreciated by those skilled in the art thatthe design of the expression vector can depend on such factors as thechoice of the host cell to be transformed, the level of expressiondesired, etc. A vector can be introduced into host cells to therebyproduce transcripts, proteins, or peptides, including fusion proteins orpeptides, encoded by nucleic acids as described herein (e.g., clusteredregularly interspersed short palindromic repeats (CRISPR) transcripts,proteins, enzymes, mutant forms thereof, fusion proteins thereof, etc.).

Advantageous vectors include lentiviruses and adeno-associated viruses,and types of such vectors can also be selected for targeting particulartypes of cells.

As used herein, the term “crRNA” or “guide RNA” or “single guide RNA” or“sgRNA” or “one or more nucleic acid components” of a Type VI CRISPR-Caslocus effector protein comprises any polynucleotide sequence havingsufficient complementarity with a target nucleic acid sequence tohybridize with the target nucleic acid sequence and directsequence-specific binding of a RNA-targeting complex to the target RNAsequence.

In certain embodiments, the CRISPR system as provided herein can makeuse of a crRNA or analogous polynucleotide comprising a guide sequence,wherein the polynucleotide is an RNA, a DNA or a mixture of RNA and DNA,and/or wherein the polynucleotide comprises one or more nucleotideanalogs. The sequence can comprise any structure, including but notlimited to a structure of a native crRNA, such as a bulge, a hairpin ora stem loop structure. In certain embodiments, the polynucleotidecomprising the guide sequence forms a duplex with a secondpolynucleotide sequence which can be an RNA or a DNA sequence.

In certain embodiments, guides of the invention comprise non-naturallyoccurring nucleic acids and/or non-naturally occurring nucleotidesand/or nucleotide analogs, and/or chemically modifications.Non-naturally occurring nucleic acids can include, for example, mixturesof naturally and non-naturally occurring nucleotides. Non-naturallyoccurring nucleotides and/or nucleotide analogs may be modified at theribose, phosphate, and/or base moiety. In an embodiment of theinvention, a guide nucleic acid comprises ribonucleotides andnon-ribonucleotides. In one such embodiment, a guide comprises one ormore ribonucleotides and one or more deoxyribonucleotides. In anembodiment of the invention, the guide comprises one or morenon-naturally occurring nucleotide or nucleotide analog such as anucleotide with phosphorothioate linkage, boranophosphate linkage, alocked nucleic acid (LNA) nucleotides comprising a methylene bridgebetween the 2′ and 4′ carbons of the ribose ring, or bridged nucleicacids (BNA). Other examples of modified nucleotides include 2′-O-methylanalogs, 2′-deoxy analogs, 2-thiouridine analogs, N6-methyladenosineanalogs, or 2′-fluoro analogs. Further examples of modified basesinclude, but are not limited to, 2-aminopurine, 5-bromo-uridine,pseudouridine (Ψ), N1-methylpseudouridine (me1Ψ),5-methoxyuridine(5moU), inosine, 7-methylguanosine. Examples of guideRNA chemical modifications include, without limitation, incorporation of2′-O-methyl (M), 2′-O-methyl 3′phosphorothioate (MS), S-constrainedethyl (cEt), or 2′-O-methyl 3′thioPACE (MSP) at one or more terminalnucleotides. Such chemically modified guide RNAs can comprise increasedstability and increased activity as compared to unmodified guide RNAs,though on-target vs. off-target specificity is not predictable. (See,Hendel, 2015, Nat Biotechnol. 33(9):985-9, doi: 10.1038/nbt.3290,published online 29 Jun. 2015; Allerson et al., J. Med. Chem. 2005,48:901-904; Bramsen et al., Front. Genet., 2012, 3:154; Deng et al.,PNAS, 2015, 112:11870-11875; Sharma et al., MedChemComm., 2014,5:1454-1471; Li et al., Nature Biomedical Engineering, 2017, 1, 0066DOI:10.1038/s41551-017-0066).

In some embodiments, the 5′ and/or 3′ end of a guide RNA is modified bya variety of functional moieties including fluorescent dyes,polyethylene glycol, cholesterol, proteins, or detection tags. (SeeKelly et al., 2016, J. Biotech. 233:74-83). In certain embodiments, aguide comprises ribonucleotides in a region that binds to a target DNAand one or more deoxyribonucleotides and/or nucleotide analogs in aregion that binds to Cas9, Cpf1, or C2c1. In an embodiment of theinvention, deoxyribonucleotides and/or nucleotide analogs areincorporated in engineered guide structures, such as, withoutlimitation, 5′ and/or 3′ end, stem-loop regions, and the seed region. Incertain embodiments, the modification is not in the 5′-handle of thestem-loop regions. Chemical modification in the 5′-handle of thestem-loop region of a guide may abolish its function (see Li, et al.,Nature Biomedical Engineering, 2017, 1:0066). In certain embodiments, atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or 75nucleotides of a guide is chemically modified. In some embodiments, 3-5nucleotides at either the 3′ or the 5′ end of a guide is chemicallymodified. In some embodiments, only minor modifications are introducedin the seed region, such as 2′-F modifications. In some embodiments,2′-F modification is introduced at the 3′ end of a guide. In certainembodiments, three to five nucleotides at the 5′ and/or the 3′ end ofthe guide are chemically modified with 2′-O-methyl (M),2′-O-methyl-3′-phosphorothioate (MS), S-constrained ethyl(cEt), or2′-O-methyl-3′-thioPACE (MSP). Such modification can enhance genomeediting efficiency (see Hendel et al., Nat. Biotechnol. (2015) 33(9):985-989). In certain embodiments, all of the phosphodiester bonds of aguide are substituted with phosphorothioates (PS) for enhancing levelsof gene disruption. In certain embodiments, more than five nucleotidesat the 5′ and/or the 3′ end of the guide are chemicially modified with2′-O-Me, 2′-F or S-constrained ethyl(cEt). Such chemically modifiedguide can mediate enhanced levels of gene disruption (see Ragdarm etal., 0215, PNAS, E7110-E7111). In an embodiment of the invention, aguide is modified to comprise a chemical moiety at its 3′ and/or 5′ end.Such moieties include, but are not limited to amine, azide, alkyne,thio, dibenzocyclooctyne (DBCO), or Rhodamine. In certain embodiment,the chemical moiety is conjugated to the guide by a linker, such as analkyl chain. In certain embodiments, the chemical moiety of the modifiedguide can be used to attach the guide to another molecule, such as DNA,RNA, protein, or nanoparticles. Such chemically modified guide can beused to identify or enrich cells generically edited by a CRISPR system(see Lee et al., eLife, 2017, 6:e25312, DOI:10.7554).

In one aspect of the invention, the guide comprises a modified crRNA forCpf1, having a 5′-handle and a guide segment further comprising a seedregion and a 3′-terminus. In some embodiments, the modified guide can beused with a Cpf1 of any one of Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1);Francisella tularensis subsp. Novicida U112 Cpf1 (FnCpf1); L. bacteriumMC2017 Cpf1 (Lb3Cpf1); Butyrivibrio proteoclasticus Cpf1 (BpCpf1);Parcubacteria bacterium GWC2011_GWC2_44_17 Cpf1 (PbCpf1);Peregrinibacteria bacterium GW2011_GWA_3310 Cpf1 (PeCpf1); Leptospirainadai Cpf1 (LiCpf1); Smithella sp. SCK08D17 Cpf1 (SsCpf1); L. bacteriumMA2020 Cpf1 (Lb2Cpf1); Porphyromonas crevioricanis Cpf1 (PcCpf1);Porphyromonas macacae Cpf1 (PmCpf1); Candidatus Methanoplasma termitumCpf1 (CMtCpf1); Eubacterium eligens Cpf1 (EeCpf1); Moraxella bovoculi237 Cpf1 (MbCpf1); Prevotella disiens Cpf1 (PdCpf1); or L. bacteriumND2006 Cpf1 (LbCpf1).

In some embodiments, the modification to the guide is a chemicalmodification, an insertion, a deletion or a split. In some embodiments,the chemical modification includes, but is not limited to, incorporationof 2′-O-methyl (M) analogs, 2′-deoxy analogs, 2-thiouridine analogs,N6-methyladenosine analogs, 2′-fluoro analogs, 2-aminopurine,5-bromo-uridine, pseudouridine (Ψ), N1-methylpseudouridine (me1Ψ),5-methoxyuridine(5moU), inosine, 7-methylguanosine,2′-O-methyl-3′-phosphorothioate (MS), S-constrained ethyl(cEt),phosphorothioate (PS), or 2′-O-methyl-3′-thioPACE (MSP). In someembodiments, the guide comprises one or more of phosphorothioatemodifications. In certain embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 25 nucleotides of theguide are chemically modified. In certain embodiments, one or morenucleotides in the seed region are chemically modified. In certainembodiments, one or more nucleotides in the 3′-terminus are chemicallymodified. In certain embodiments, none of the nucleotides in the5′-handle is chemically modified. In some embodiments, the chemicalmodification in the seed region is a minor modification, such asincorporation of a 2′-fluoro analog. In a specific embodiment, onenucleotide of the seed region is replaced with a 2′-fluoro analog. Insome embodiments, 5 or 10 nucleotides in the 3′-terminus are chemicallymodified. Such chemical modifications at the 3′-terminus of the Cpf1CrRNA improve gene cutting efficiency (see Li, et al., Nature BiomedicalEngineering, 2017, 1:0066). In a specific embodiment, 5 nucleotides inthe 3′-terminus are replaced with 2′-fluoro analogues. In a specificembodiment, 10 nucleotides in the 3′-terminus are replaced with2′-fluoro analogues. In a specific embodiment, 5 nucleotides in the3′-terminus are replaced with 2′-O-methyl (M) analogs.

In some embodiments, the loop of the 5′-handle of the guide is modified.In some embodiments, the loop of the 5′-handle of the guide is modifiedto have a deletion, an insertion, a split, or chemical modifications. Incertain embodiments, the loop comprises 3, 4, or 5 nucleotides. Incertain embodiments, the loop comprises the sequence of UCUU, UUUU,UAUU, or UGUU.

In one aspect, the guide comprises portions that are chemically linkedor conjugated via a non-phosphodiester bond. In one aspect, the guidecomprises, in non-limiting examples, a tracr sequence and a tracr matesequence portion or a direct repeat and a targeting sequence portionthat are chemically linked or conjugated via a non-nucleotide loop. Insome embodiments, the portions are joined via a non-phosphodiestercovalent linker. Examples of the covalent linker include but are notlimited to a chemical moiety selected from the group consisting ofcarbamates, ethers, esters, amides, imines, amidines, aminotrizines,hydrozone, disulfides, thioethers, thioesters, phosphorothioates,phosphorodithioates, sulfonamides, sulfonates, fulfones, sulfoxides,ureas, thioureas, hydrazide, oxime, triazole, photolabile linkages, C—Cbond forming groups such as Diels-Alder cyclo-addition pairs orring-closing metathesis pairs, and Michael reaction pairs.

In some embodiments, portions of the guide are first synthesized usingthe standard phosphoramidite synthetic protocol (Herdewijn, P., ed.,Methods in Molecular Biology Col 288, Oligonucleotide Synthesis: Methodsand Applications, Humana Press, New Jersey (2012)). In some embodiments,the non-targeting guide portions can be functionalized to contain anappropriate functional group for ligation using the standard protocolknown in the art (Hermanson, G. T., Bioconjugate Techniques, AcademicPress (2013)). Examples of functional groups include, but are notlimited to, hydroxyl, amine, carboxylic acid, carboxylic acid halide,carboxylic acid active ester, aldehyde, carbonyl, chlorocarbonyl,imidazolylcarbonyl, hydrozide, semicarbazide, thio semicarbazide, thiol,maleimide, haloalkyl, sufonyl, ally, propargyl, diene, alkyne, andazide. Once a non-targeting portions of a guide is functionalized, acovalent chemical bond or linkage can be formed between the twooligonucleotides. Examples of chemical bonds include, but are notlimited to, those based on carbamates, ethers, esters, amides, imines,amidines, aminotrizines, hydrozone, disulfides, thioethers, thioesters,phosphorothioates, phosphorodithioates, sulfonamides, sulfonates,fulfones, sulfoxides, ureas, thioureas, hydrazide, oxime, triazole,photolabile linkages, C—C bond forming groups such as Diels-Aldercyclo-addition pairs or ring-closing metathesis pairs, and Michaelreaction pairs.

In some embodiments, one or more portions of a guide can be chemicallysynthesized. In some embodiments, the chemical synthesis uses automated,solid-phase oligonucleotide synthesis machines with 2′-acetoxyethylorthoester (2′-ACE) (Scaringe et al., J. Am. Chem. Soc. (1998) 120:11820-11821; Scaringe, Methods Enzymol. (2000) 317: 3-18) or2′-thionocarbamate (2′-TC) chemistry (Dellinger et al., J. Am. Chem.Soc. (2011) 133: 11540-11546; Hendel et al., Nat. Biotechnol. (2015)33:985-989).

In some embodiments, the guide portions can be covalently linked usingvarious bioconjugation reactions, loops, bridges, and non-nucleotidelinks via modifications of sugar, internucleotide phosphodiester bonds,purine and pyrimidine residues. Sletten et al., Angew. Chem. Int. Ed.(2009) 48:6974-6998; Manoharan, M. Curr. Opin. Chem. Biol. (2004) 8:570-9; Behlke et al., Oligonucleotides (2008) 18: 305-19; Watts, et al.,Drug. Discov. Today (2008) 13: 842-55; Shukla, et al., ChemMedChem(2010) 5: 328-49.

In some embodiments, the guide portions can be covalently linked usingclick chemistry. In some embodiments, guide portions can be covalentlylinked using a triazole linker. In some embodiments, guide portions canbe covalently linked using Huisgen 1,3-dipolar cycloaddition reactioninvolving an alkyne and azide to yield a highly stable triazole linker(He et al., ChemBioChem (2015) 17: 1809-1812; WO 2016/186745). In someembodiments, guide portions are covalently linked by ligating a5′-hexyne portion and a 3′-azide portion. In some embodiments, either orboth of the 5′-hexyne guide portion and a 3′-azide guide portion can beprotected with 2′-acetoxyethyl orthoester (2′-ACE) group, which can besubsequently removed using Dharmacon protocol (Scaringe et al., J. Am.Chem. Soc. (1998) 120: 11820-11821; Scaringe, Methods Enzymol. (2000)317: 3-18).

In some embodiments, guide portions can be covalently linked via alinker (e.g., a non-nucleotide loop) that comprises a moiety such asspacers, attachments, bioconjugates, chromophores, reporter groups, dyelabeled RNAs, and non-naturally occurring nucleotide analogues. Morespecifically, suitable spacers for purposes of this invention include,but are not limited to, polyethers (e.g., polyethylene glycols,polyalcohols, polypropylene glycol or mixtures of ethylene and propyleneglycols), polyamines group (e.g., spennine, spermidine and polymericderivatives thereof), polyesters (e.g., poly(ethyl acrylate)),polyphosphodiesters, alkylenes, and combinations thereof. Suitableattachments include any moiety that can be added to the linker to addadditional properties to the linker, such as but not limited to,fluorescent labels. Suitable bioconjugates include, but are not limitedto, peptides, glycosides, lipids, cholesterol, phospholipids, diacylglycerols and dialkyl glycerols, fatty acids, hydrocarbons, enzymesubstrates, steroids, biotin, digoxigenin, carbohydrates,polysaccharides. Suitable chromophores, reporter groups, and dye-labeledRNAs include, but are not limited to, fluorescent dyes such asfluorescein and rhodamine, chemiluminescent, electrochemiluminescent,and bioluminescent marker compounds. The design of example linkersconjugating two RNA components are also described in WO 2004/015075.

The linker (e.g., a non-nucleotide loop) can be of any length. In someembodiments, the linker has a length equivalent to about 0-16nucleotides. In some embodiments, the linker has a length equivalent toabout 0-8 nucleotides. In some embodiments, the linker has a lengthequivalent to about 0-4 nucleotides. In some embodiments, the linker hasa length equivalent to about 2 nucleotides. Example linker design isalso described in WO 2011/008730.

In some embodiments, the degree of complementarity, when optimallyaligned using a suitable alignment algorithm, is about or more thanabout 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimalalignment may be determined with the use of any suitable algorithm foraligning sequences, non-limiting example of which include theSmith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithmsbased on the Burrows-Wheeler Transform (e.g., the Burrows WheelerAligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies;available at www.novocraft.com), ELAND (Illumina, San Diego, CA), SOAP(available at soap.genomics.org.cn), and Maq (available atmaq.sourceforge.net). The ability of a guide sequence (within aRNA-targeting guide RNA or crRNA) to direct sequence-specific binding ofa nucleic acid-targeting complex to a target nucleic acid sequence maybe assessed by any suitable assay. For example, the components of aRNA-targeting CRISPR Cas13b system sufficient to form a nucleicacid-targeting complex, including the guide sequence to be tested, maybe provided to a host cell having the corresponding target nucleic acidsequence, such as by transfection with vectors encoding the componentsof the nucleic acid-targeting complex, followed by an assessment ofpreferential targeting (e.g., cleavage) within the target nucleic acidsequence, such as by Surveyor assay as described herein. Similarly,cleavage of a target nucleic acid sequence may be evaluated in a testtube by providing the target nucleic acid sequence, components of anucleic acid-targeting complex, including the guide sequence to betested and a control guide sequence different from the test guidesequence, and comparing binding or rate of cleavage at the targetsequence between the test and control guide sequence reactions. Otherassays are possible, and will occur to those skilled in the art. A guidesequence, and hence a RNA-targeting guide RNA or crRNA may be selectedto target any target nucleic acid sequence. The target sequence may beDNA. The target sequence may be any RNA sequence. In some embodiments,the target sequence may be a sequence within a RNA molecule selectedfrom the group consisting of messenger RNA (mRNA), pre-mRNA, ribosomalRNA (rRNA), transfer RNA (tRNA), micro-RNA (miRNA), small interferingRNA (siRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA),double stranded RNA (dsRNA), non coding RNA (ncRNA), long non-coding RNA(lncRNA), and small cytoplasmatic RNA (scRNA). In some preferredembodiments, the target sequence may be a sequence within a RNA moleculeselected from the group consisting of mRNA, pre-mRNA, and rRNA. In somepreferred embodiments, the target sequence may be a sequence within aRNA molecule selected from the group consisting of ncRNA, and lncRNA. Insome more preferred embodiments, the target sequence may be a sequencewithin an mRNA molecule or a pre-mRNA molecule.

In some embodiments, a RNA-targeting guide RNA or crRNA is selected toreduce the degree secondary structure within the RNA-targeting guide RNAor crRNA. In some embodiments, about or less than about 75%, 50%, 40%,30%, 25%, 20%, 15%, 10%, 5%, 1%, or fewer of the nucleotides of theRNA-targeting guide RNA participate in self-complementary base pairingwhen optimally folded. Optimal folding may be determined by any suitablepolynucleotide folding algorithm. Some programs are based on calculatingthe minimal Gibbs free energy. An example of one such algorithm ismFold, as described by Zuker and Stiegler (Nucleic Acids Res. 9 (1981),133-148). Another example folding algorithm is the online webserver RNAfold, developed at Institute for Theoretical Chemistry at the Universityof Vienna, using the centroid structure prediction algorithm (see e.g.,A. R. Gruber et al., 2008, Cell 106(1): 23-24; and PA Carr and GMChurch, 2009, Nature Biotechnology 27(12): 1151-62).

In certain embodiments, a guide RNA or crRNA may comprise, consistessentially of, or consist of a direct repeat (DR) sequence and a guidesequence or spacer sequence. In certain embodiments, the guide RNA orcrRNA may comprise, consist essentially of, or consist of a directrepeat sequence fused or linked to a guide sequence or spacer sequence.In certain embodiments, the direct repeat sequence may be locatedupstream (i.e., 5′) from the guide sequence or spacer sequence. In otherembodiments, the direct repeat sequence may be located downstream (i.e.,3′) from the guide sequence or spacer sequence. In other embodiments,multiple DRs (such as dual DRs) may be present.

In certain embodiments, the crRNA comprises a stem loop, preferably asingle stem loop. In certain embodiments, the direct repeat sequenceforms a stem loop, preferably a single stem loop.

In certain embodiments, the spacer length of the guide RNA is from 15 to35 nt. In certain embodiments, the spacer length of the guide RNA is atleast 15 nucleotides. In certain embodiments, the spacer length is from15 to 17 nt, e.g., 15, 16, or 17 nt, from 17 to 20 nt, e.g., 17, 18, 19,or 20 nt, from 20 to 24 nt, e.g., 20, 21, 22, 23, or 24 nt, from 23 to25 nt, e.g., 23, 24, or 25 nt, from 24 to 27 nt, e.g., 24, 25, 26, or 27nt, from 27-30 nt, e.g., 27, 28, 29, or 30 nt, from 30-35 nt, e.g., 30,31, 32, 33, 34, or 35 nt, or 35 nt or longer.

The “tracrRNA” sequence or analogous terms includes any polynucleotidesequence that has sufficient complementarity with a crRNA sequence tohybridize. In general, degree of complementarity is with reference tothe optimal alignment of the sca sequence and tracr sequence, along thelength of the shorter of the two sequences. Optimal alignment may bedetermined by any suitable alignment algorithm, and may further accountfor secondary structures, such as self-complementarity within either thesca sequence or tracr sequence. In some embodiments, the degree ofcomplementarity between the tracr sequence and sca sequence along thelength of the shorter of the two when optimally aligned is about or morethan about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99%, orhigher. In certain embodiments, the tracrRNA may not be required.Indeed, the Cas13b effector protein from Bergeyella zoohelcum andorthologs thereof do not require a tracrRNA to ensure cleavage of an RNAtarget.

In further detail, the assay is as follows for a RNA target, providedthat a PAM sequence is required to direct recognition. Two E. colistrains are used in this assay. One carries a plasmid that encodes theendogenous effector protein locus from the bacterial strain. The otherstrain carries an empty plasmid (e.g. pACYC184, control strain). Allpossible 7 or 8 bp PAM sequences are presented on an antibioticresistance plasmid (pUC19 with ampicillin resistance gene). The PAM islocated next to the sequence of proto-spacer 1 (the RNA target to thefirst spacer in the endogenous effector protein locus). Two PAMlibraries were cloned. One has a 8 random bp 5′ of the proto-spacer(e.g. total of 65536 different PAM sequences=complexity). The otherlibrary has 7 random bp 3′ of the proto-spacer (e.g. total complexity is16384 different PAMs). Both libraries were cloned to have in average 500plasmids per possible PAM. Test strain and control strain weretransformed with 5′PAM and 3′PAM library in separate transformations andtransformed cells were plated separately on ampicillin plates.Recognition and subsequent cutting/interference with the plasmid rendersa cell vulnerable to ampicillin and prevents growth. Approximately 12 hafter transformation, all colonies formed by the test and controlstrains where harvested and plasmid RNA was isolated. Plasmid RNA wasused as template for PCR amplification and subsequent deep sequencing.Representation of all PAMs in the untransformed libraries showed theexpected representation of PAMs in transformed cells. Representation ofall PAMs found in control strains showed the actual representation.Representation of all PAMs in test strain showed which PAMs are notrecognized by the enzyme and comparison to the control strain allowsextracting the sequence of the depleted PAM. In particular embodiments,the cleavage, such as the RNA cleavage is not PAM dependent. Indeed, forthe Bergeyella zoohelcum effector protein and its orthologs, RNA targetcleavage appears to be PAM independent, and hence the Table 1A or Table1B Cas13b of the invention may act in a PAM independent fashion.

For minimization of toxicity and off-target effect, it will be importantto control the concentration of RNA-targeting guide RNA delivered.Optimal concentrations of nucleic acid-targeting guide RNA can bedetermined by testing different concentrations in a cellular or nonhuman eukaryote animal model and using deep sequencing the analyze theextent of modification at potential off-target genomic loci. Theconcentration that gives the highest level of on-target modificationwhile minimizing the level of off-target modification should be chosenfor in vivo delivery. The RNA-targeting system is derived advantageouslyfrom a CRISPR-Cas13b system. In some embodiments, one or more elementsof a RNA-targeting system is derived from a particular organismcomprising an endogenous RNA-targeting system of a Table 1A or Table 1BCas13b effector protein system as herein-discussed.

The terms “orthologue” (also referred to as “ortholog” herein) and“homologue” (also referred to as “homolog” herein) are well known in theart. By means of further guidance, a “homologue” of a protein as usedherein is a protein of the same species which performs the same or asimilar function as the protein it is a homologue of Homologous proteinsmay but need not be structurally related, or are only partiallystructurally related. An “orthologue” of a protein as used herein is aprotein of a different species which performs the same or a similarfunction as the protein it is an orthologue of Orthologous proteins maybut need not be structurally related, or are only partially structurallyrelated. In particular embodiments, the homologue or orthologue of aCas13b protein as referred to herein has a sequence homology or identityof at least 80%, more preferably at least 85%, even more preferably atleast 90%, such as for instance at least 95% with a Cas13b effectorprotein set forth in Table 1A or Table 1B, below. In a preferredembodiment, the Cas13b effector protein may be of or from an organismidentified in Table 1A or Table 1B or the genus to which the organismbelongs.

It has been found that a number of Cas13b orthologs are characterized bycommon motifs. Accordingly, in particular embodiments, the Cas13beffector protein is a protein comprising a sequence having at least 70%sequence identity with one or more of the sequences consisting ofDKHXFGAFLNLARHN (SEQ ID NO: 22), GLLFFVSLFLDK (SEQ ID NO: 23), SKIXGFK(SEQ ID NO: 24), DMLNELXRCP (SEQ ID NO: 25), RXZDRFPYFALRYXD (SEQ ID NO:26) and LRFQVBLGXY (SEQ ID NO: 27). In further particular embodiments,the Cas13b effector protein comprises a sequence having at least 70%sequence identity at least 2, 3, 4, 5 or all 6 of these sequences. Infurther particular embodiments, the sequence identity with thesesequences is at least 75%, 80%, 85%, 90%, 95% or 100%. In furtherparticular embodiments, the Cas13b effector protein is a proteincomprising a sequence having 100% sequence identity with GLLFFVSLFL (SEQID NO: 28) and RHQXRFPYF (SEQ ID NO: 29). In further particularembodiments, the Cas13b effector is a Cas13b effector protein comprisinga sequence having 100% sequence identity with RHQDRFPY (SEQ ID NO: 30).

In particular embodiments, the Cas13b effector protein is a Cas13beffector protein having at least 65%, preferably at least 70%, 75%, 80%,85%, 90%, 95% or more sequence identity with a Cas13b protein fromPrevotella buccae, Porphyromonas gingivalis, Prevotella saccharolytica,Riemerella anatipestifer. In further particular embodiments, the Cas13beffector is selected from the Cas13b protein from Bacteroides pyogenes,Prevotella sp. MA2016, Riemerella anatipestifer, Porphyromonas gulae,Porphyromonas gingivalis, and Porphyromonas sp. COT-052OH4946.

It will be appreciated that orthologs of a Table 1A or Table 1B Cas13benzyme that can be within the invention can include a chimeric enzymecomprising a fragment of a Table 1A or Table 1B Cas13b enzyme multipleorthologs. Examples of such orthologs are described elsewhere herein. Achimeric enzyme may comprise a fragment of a Table 1A or Table 1B Cas13benzyme and a fragment from another CRISPR enzyme, such as an ortholog ofa Table 1A or Table 1B Cas13b enzyme of an organism which includes butis not limited to Bergeyella, Prevotella, Porphyromonas, Bacteroides,Alistipes, Riemerella, Myroides, Flavobacterium, Capnocytophaga,Chryseobacterium, Phaeodactylibacter, Paludibacter or Psychroflexus. Achimeric enzyme can comprise a first fragment and a second fragment, andthe fragments, wherein one of the first and second a fragment is of orfrom a Table 1A or Table 1B Cas13b enzyme and the other fragment is ofor from a CRISPR enzyme ortholog of a different species.

In embodiments, the Cas13b RNA-targeting Cas13b effector proteinsreferred to herein also encompasses a functional variant of the effectorprotein or a homologue or an orthologue thereof. A “functional variant”of a protein as used herein refers to a variant of such protein whichretains at least partially the activity of that protein. Functionalvariants may include mutants (which may be insertion, deletion, orreplacement mutants), including polymorphs, etc., including as discussedherein in conjunction with Table 1A or Table 1B. Also included withinfunctional variants are fusion products of such protein with another,usually unrelated, nucleic acid, protein, polypeptide or peptide.Functional variants may be naturally occurring or may be man-made. In anembodiment, nucleic acid molecule(s) encoding the Cas13b RNA-targetingeffector proteins, or an ortholog or homolog thereof, may becodon-optimized for expression in an eukaryotic cell. A eukaryote can beas herein discussed. Nucleic acid molecule(s) can be engineered ornon-naturally occurring.

In an embodiment, the Cas13b RNA-targeting effector protein or anortholog or homolog thereof, may comprise one or more mutations. Themutations may be artificially introduced mutations and may include butare not limited to one or more mutations in a catalytic domain, e.g.,one or more mutations are introduced into one or more of the HEPNdomains.

In an embodiment, the Cas13b protein or an ortholog or homolog thereof,may be used as a generic nucleic acid binding protein with fusion to orbeing operably linked to a functional domain. Exemplary functionaldomains may include but are not limited to translational initiator,translational activator, translational repressor, nucleases, inparticular ribonucleases, a spliceosome, beads, a lightinducible/controllable domain or a chemically inducible/controllabledomain.

In some embodiments, the unmodified RNA-targeting effector protein(Cas13b) may have cleavage activity. In some embodiments, Cas13b maydirect cleavage of one or two nucleic acid strands at the location of ornear a target sequence, such as within the target sequence and/or withinthe complement of the target sequence or at sequences associated withthe target sequence, e.g., within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or lastnucleotide of a target sequence. In some embodiments, the Cas13b proteinmay direct more than one cleavage (such as one, two three, four, five,or more cleavages) of one or two strands within the target sequenceand/or within the complement of the target sequence or at sequencesassociated with the target sequence and/or within about 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs fromthe first or last nucleotide of a target sequence. In some embodiments,the cleavage may be blunt, i.e., generating blunt ends. In someembodiments, the cleavage may be staggered, i.e., generating stickyends. In some embodiments, a vector encodes a nucleic acid-targetingCas13b protein that may be mutated with respect to a correspondingwild-type enzyme such that the mutated nucleic acid-targeting Cas13bprotein lacks the ability to cleave one or two strands of a targetpolynucleotide containing a target sequence, e.g., alteration ormutation in a HEPN domain to produce a mutated Cas13b substantiallylacking all RNA cleavage activity, e.g., the RNA cleavage activity ofthe mutated enzyme is about no more than 25%, 10%, 5%, 1%, 0.1%, 0.01%,or less of the nucleic acid cleavage activity of the non-mutated form ofthe enzyme; an example can be when the nucleic acid cleavage activity ofthe mutated form is nil or negligible as compared with the non-mutatedform. By derived, Applicants mean that the derived enzyme is largelybased, in the sense of having a high degree of sequence homology with, awildtype enzyme, but that it has been mutated (modified) in some way asknown in the art or as described herein.

Typically, in the context of an endogenous RNA-targeting system,formation of a RNA-targeting complex (comprising a guide RNA or crRNAhybridized to a target sequence and complexed with one or moreRNA-targeting effector proteins) results in cleavage of RNA strand(s) inor near (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or morebase pairs from) the target sequence. As used herein the term“sequence(s) associated with a target locus of interest” refers tosequences near the vicinity of the target sequence (e.g. within 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from the targetsequence, wherein the target sequence is comprised within a target locusof interest).

An example of a codon optimized sequence, is in this instance a sequenceoptimized for expression in a eukaryote, e.g., humans (i.e. beingoptimized for expression in humans), or for another eukaryote, animal ormammal as herein discussed; see, e.g., SaCas9 human codon optimizedsequence in WO 2014/093622 (PCT/US2013/074667) as an example of a codonoptimized sequence (from knowledge in the art and this disclosure, codonoptimizing coding nucleic acid molecule(s), especially as to effectorprotein (e.g., Cas13b) is within the ambit of the skilled artisan).Whilst this is preferred, it will be appreciated that other examples arepossible and codon optimization for a host species other than human, orfor codon optimization for specific organs is known. In someembodiments, an enzyme coding sequence encoding a RNA-targeting Cas13bprotein is codon optimized for expression in particular cells, such aseukaryotic cells. The eukaryotic cells may be those of or derived from aparticular organism, such as a mammal, including but not limited tohuman, or non-human eukaryote or animal or mammal as herein discussed,e.g., mouse, rat, rabbit, dog, livestock, or non-human mammal orprimate. In some embodiments, processes for modifying the germ linegenetic identity of human beings and/or processes for modifying thegenetic identity of animals which are likely to cause them sufferingwithout any substantial medical benefit to man or animal, and alsoanimals resulting from such processes, may be excluded. In general,codon optimization refers to a process of modifying a nucleic acidsequence for enhanced expression in the host cells of interest byreplacing at least one codon (e.g., about or more than about 1, 2, 3, 4,5, 10, 15, 20, 25, 50, or more codons) of the native sequence withcodons that are more frequently or most frequently used in the genes ofthat host cell while maintaining the native amino acid sequence. Variousspecies exhibit particular bias for certain codons of a particular aminoacid. Codon bias (differences in codon usage between organisms) oftencorrelates with the efficiency of translation of messenger RNA (mRNA),which is in turn believed to be dependent on, among other things, theproperties of the codons being translated and the availability ofparticular transfer RNA (tRNA) molecules. The predominance of selectedtRNAs in a cell is generally a reflection of the codons used mostfrequently in peptide synthesis. Accordingly, genes can be tailored foroptimal gene expression in a given organism based on codon optimization.Codon usage tables are readily available, for example, at the “CodonUsage Database” available at www.kazusa.or.jp/codon/and these tables canbe adapted in a number of ways. See Nakamura, Y., et al. “Codon usagetabulated from the international DNA sequence databases: status for theyear 2000” Nucl. Acids Res. 28:292 (2000). Computer algorithms for codonoptimizing a particular sequence for expression in a particular hostcell are also available, such as Gene Forge (Aptagen; Jacobus, PA), arealso available. In some embodiments, one or more codons (e.g., 1, 2, 3,4, 5, 10, 15, 20, 25, 50, or more, or all codons) in a sequence encodinga DNA/RNA-targeting Cas protein corresponds to the most frequently usedcodon for a particular amino acid.

The (i) Cas13b or nucleic acid molecule(s) encoding it or (ii) crRNA canbe delivered separately; and advantageously at least one or both of oneof (i) and (ii), e.g., an assembled complex is delivered via a particleor nanoparticle complex. RNA-targeting effector protein mRNA can bedelivered prior to the RNA-targeting guide RNA or crRNA to give time fornucleic acid-targeting effector protein to be expressed. RNA-targetingeffector protein (Cas13b) mRNA might be administered 1-12 hours(preferably around 2-6 hours) prior to the administration ofRNA-targeting guide RNA or crRNA. Alternatively, RNA-targeting effectorprotein mRNA and RNA-targeting guide RNA or crRNA can be administeredtogether. Advantageously, a second booster dose of guide RNA or crRNAcan be administered 1-12 hours (preferably around 2-6 hours) after theinitial administration of RNA-targeting effector (Cas13b) proteinmRNA+guide RNA. Additional administrations of RNA-targeting effectorprotein mRNA and/or guide RNA or crRNA might be useful to achieve themost efficient levels of genome modification.

In one aspect, the invention provides methods for using one or moreelements of a RNA-targeting system. The RNA-targeting complex of theinvention provides an effective means for modifying a target RNA singleor double stranded, linear or super-coiled. The RNA-targeting complex ofthe invention has a wide variety of utility including modifying (e.g.,deleting, inserting, translocating, inactivating, activating) a targetRNA in a multiplicity of cell types. As such the RNA-targeting complexof the invention has a broad spectrum of applications in, e.g., genetherapy, drug screening, disease diagnosis, and prognosis. An exemplaryRNA-targeting complex comprises a RNA-targeting effector proteincomplexed with a guide RNA or crRNA hybridized to a target sequencewithin the target locus of interest.

In one embodiment, this invention provides a method of cleaving a targetRNA. The method may comprise modifying a target RNA using aRNA-targeting complex that binds to the target RNA and effect cleavageof said target RNA. In an embodiment, the RNA-targeting complex of theinvention, when introduced into a cell, may create a break (e.g., asingle or a double strand break) in the RNA sequence. For example, themethod can be used to cleave a disease RNA in a cell. For example, anexogenous RNA template comprising a sequence to be integrated flanked byan upstream sequence and a downstream sequence may be introduced into acell. The upstream and downstream sequences share sequence similaritywith either side of the site of integration in the RNA. Where desired, adonor RNA can be mRNA. The exogenous RNA template comprises a sequenceto be integrated (e.g., a mutated RNA). The sequence for integration maybe a sequence endogenous or exogenous to the cell. Examples of asequence to be integrated include RNA encoding a protein or a non-codingRNA (e.g., a microRNA). Thus, the sequence for integration may beoperably linked to an appropriate control sequence or sequences.Alternatively, the sequence to be integrated may provide a regulatoryfunction. The upstream and downstream sequences in the exogenous RNAtemplate are selected to promote recombination between the RNA sequenceof interest and the donor RNA. The upstream sequence is a RNA sequencethat shares sequence similarity with the RNA sequence upstream of thetargeted site for integration. Similarly, the downstream sequence is aRNA sequence that shares sequence similarity with the RNA sequencedownstream of the targeted site of integration. The upstream anddownstream sequences in the exogenous RNA template can have 75%, 80%,85%, 90%, 95%, or 100% sequence identity with the targeted RNA sequence.Preferably, the upstream and downstream sequences in the exogenous RNAtemplate have about 95%, 96%, 97%, 98%, 99%, or 100% sequence identitywith the targeted RNA sequence. In some methods, the upstream anddownstream sequences in the exogenous RNA template have about 99% or100% sequence identity with the targeted RNA sequence. An upstream ordownstream sequence may comprise from about 20 bp to about 2500 bp, forexample, about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000,1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200,2300, 2400, or 2500 bp. In some methods, the exemplary upstream ordownstream sequence have about 200 bp to about 2000 bp, about 600 bp toabout 1000 bp, or more particularly about 700 bp to about 1000 bp. Insome methods, the exogenous RNA template may further comprise a marker.Such a marker may make it easy to screen for targeted integrations.Examples of suitable markers include restriction sites, fluorescentproteins, or selectable markers. The exogenous RNA template of theinvention can be constructed using recombinant techniques (see, forexample, Sambrook et al., 2001 and Ausubel et al., 1996). In a methodfor modifying a target RNA by integrating an exogenous RNA template, abreak (e.g., double or single stranded break in double or singlestranded RNA) is introduced into the RNA sequence by the nucleicacid-targeting complex, the break is repaired via homologousrecombination with an exogenous RNA template such that the template isintegrated into the RNA target. The presence of a double-stranded breakfacilitates integration of the template. In other embodiments, thisinvention provides a method of modifying expression of a RNA in aeukaryotic cell. The method comprises increasing or decreasingexpression of a target polynucleotide by using a nucleic acid-targetingcomplex that binds to the DNA or RNA (e.g., mRNA or pre-mRNA). In somemethods, a target RNA can be inactivated to effect the modification ofthe expression in a cell. For example, upon the binding of aRNA-targeting complex to a target sequence in a cell, the target RNA isinactivated such that the sequence is not translated, the coded proteinis not produced, or the sequence does not function as the wild-typesequence does. For example, a protein or microRNA coding sequence may beinactivated such that the protein or microRNA or pre-microRNA transcriptis not produced. The target RNA of a RNA-targeting complex can be anyRNA endogenous or exogenous to the eukaryotic cell. For example, thetarget RNA can be a RNA residing in the nucleus of the eukaryotic cell.The target RNA can be a sequence (e.g., mRNA or pre-mRNA) coding a geneproduct (e.g., a protein) or a non-coding sequence (e.g., ncRNA, lncRNA,tRNA, or rRNA). Examples of target RNA include a sequence associatedwith a signaling biochemical pathway, e.g., a signaling biochemicalpathway-associated RNA. Examples of target RNA include a diseaseassociated RNA. A “disease-associated” RNA refers to any RNA which isyielding translation products at an abnormal level or in an abnormalform in cells derived from a disease-affected tissues compared withtissues or cells of a non disease control. It may be a RNA transcribedfrom a gene that becomes expressed at an abnormally high level; it maybe a RNA transcribed from a gene that becomes expressed at an abnormallylow level, where the altered expression correlates with the occurrenceand/or progression of the disease. A disease-associated RNA also refersto a RNA transcribed from a gene possessing mutation(s) or geneticvariation that is directly responsible or is in linkage disequilibriumwith a gene(s) that is responsible for the etiology of a disease. Thetranslated products may be known or unknown, and may be at a normal orabnormal level. The target RNA of a RNA-targeting complex can be any RNAendogenous or exogenous to the eukaryotic cell. For example, the targetRNA can be a RNA residing in the nucleus of the eukaryotic cell. Thetarget RNA can be a sequence (e.g., mRNA or pre-mRNA) coding a geneproduct (e.g., a protein) or a non-coding sequence (e.g., ncRNA, lncRNA,tRNA, or rRNA).

In some embodiments, the method may comprise allowing a RNA-targetingcomplex to bind to the target RNA to effect cleavage of said target RNAthereby modifying the target RNA, wherein the RNA-targeting complexcomprises a nucleic acid-targeting effector (Cas13b) protein complexedwith a guide RNA or crRNA hybridized to a target sequence within saidtarget RNA. In one aspect, the invention provides a method of modifyingexpression of RNA in a eukaryotic cell. In some embodiments, the methodcomprises allowing a RNA-targeting complex to bind to the RNA such thatsaid binding results in increased or decreased expression of said RNA;wherein the RNA-targeting complex comprises a nucleic acid-targetingeffector (Cas13b) protein complexed with a guide RNA. Methods ofmodifying a target RNA can be in a eukaryotic cell, which may be invivo, ex vivo or in vitro. In some embodiments, the method comprisessampling a cell or population of cells from a human or non-human animal,and modifying the cell or cells. Culturing may occur at any stage exvivo. The cell or cells may even be re-introduced into the non-humananimal or plant. For re-introduced cells it is particularly preferredthat the cells are stem cells.

The use of two different aptamers (each associated with a distinctRNA-targeting guide RNAs) allows an activator-adaptor protein fusion anda repressor-adaptor protein fusion to be used, with differentRNA-targeting guide RNAs or crRNAs, to activate expression of RNA,whilst repressing another. They, along with their different guide RNAsor crRNAs can be administered together, or substantially together, in amultiplexed approach. A large number of such modified RNA-targetingguide RNAs or crRNAs can be used all at the same time, for example 10 or20 or 30 and so forth, whilst only one (or at least a minimal number) ofeffector protein (Cas13b) molecules need to be delivered, as acomparatively small number of effector protein molecules can be usedwith a large number modified guides. The adaptor protein may beassociated (preferably linked or fused to) one or more activators or oneor more repressors. For example, the adaptor protein may be associatedwith a first activator and a second activator. The first and secondactivators may be the same, but they are preferably differentactivators. Three or more or even four or more activators (orrepressors) may be used, but package size may limit the number beinghigher than 5 different functional domains. Linkers are preferably used,over a direct fusion to the adaptor protein, where two or morefunctional domains are associated with the adaptor protein. Suitablelinkers might include the GlySer linker.

It is also envisaged that the RNA-targeting effector protein-guide RNAcomplex as a whole may be associated with two or more functionaldomains. For example, there may be two or more functional domainsassociated with the RNA-targeting effector protein, or there may be twoor more functional domains associated with the guide RNA or crRNA (viaone or more adaptor proteins), or there may be one or more functionaldomains associated with the RNA-targeting effector protein and one ormore functional domains associated with the guide RNA or crRNA (via oneor more adaptor proteins).

The fusion between the adaptor protein and the activator or repressormay include a linker. For example, GlySer linkers GGGS can be used. Theycan be used in repeats of 3 ((GGGGS)₃) or 6, 9 or even 12 or more, toprovide suitable lengths, as required. Linkers can be used between theguide RNAs and the functional domain (activator or repressor), orbetween the nucleic acid-targeting effector protein and the functionaldomain (activator or repressor). The linkers the user to engineerappropriate amounts of “mechanical flexibility”.

Cas13b Effector Protein Complexes can Deliver Functional Effectors

Unlike CRISPR-Cas13b-mediated knockout, which eliminates expression bymutating at the RNA level, CRISPR-Cas13b knockdown allows for temporaryreduction of gene expression through the use of artificial transcriptionfactors, e.g., via mutating residues in cleavage domain(s) of the Cas13bprotein results in the generation of a catalytically inactive Cas13bprotein. A catalytically inactive Cas13b complexes with a guide RNA orcrRNA and localizes to the RNA sequence specified by that guide RNA's orcrRNA's targeting domain, however, it does not cleave the target. Fusionof the inactive Cas13b protein to an effector domain, e.g., atranscription repression domain, enables recruitment of the effector toany site specified by the guide RNA.

Optimized Functional RNA Targeting Systems

In an aspect the invention thus provides a system for specific deliveryof functional components to the RNA environment. This can be ensuredusing the CRISPR systems comprising the RNA targeting effector proteinsof the present invention (Table 1A or Table 1B Cas13b) which allowspecific targeting of different components to RNA. More particularlysuch components include activators or repressors, such as activators orrepressors of RNA translation, degradation, etc.

According to one aspect the invention provides non-naturally occurringor engineered composition comprising a guide RNA or crRNA comprising aguide sequence capable of hybridizing to a target sequence of interestin a cell, wherein the guide RNA or crRNA is modified by the insertionof one or more distinct RNA sequence(s) that bind an adaptor protein. Inparticular embodiments, the RNA sequences may bind to two or moreadaptor proteins (e.g. aptamers), and wherein each adaptor protein isassociated with one or more functional domains. When there is more thanone functional domain, the functional domains can be same or different,e.g., two of the same or two different activators or repressors. In anaspect the invention provides a herein-discussed composition, whereinthe one or more functional domains are attached to the RNA targetingenzyme so that upon binding to the target RNA the functional domain isin a spatial orientation allowing for the functional domain to functionin its attributed function; In an aspect the invention provides aherein-discussed composition, wherein the composition comprises aCRISPR-Cas13b complex having at least three functional domains, at leastone of which is associated with the RNA targeting enzyme and at leasttwo of which are associated with the gRNA or crRNA.

Delivery of the Cas13b Effector Protein Complex or Components Thereof

Through this disclosure and the knowledge in the art, TALEs, CRISPR-Cassystems, or components thereof or nucleic acid molecules thereof(including, for instance HDR template) or nucleic acid moleculesencoding or providing components thereof may be delivered by a deliverysystem herein described both generally and in detail.

Vector delivery, e.g., plasmid, viral delivery: The CRISPR enzyme,and/or any of the present RNAs, for instance a guide RNA, can bedelivered using any suitable vector, e.g., plasmid or viral vectors,such as adeno associated virus (AAV), lentivirus, adenovirus or otherviral vector types, or combinations thereof. Effector proteins and oneor more guide RNAs can be packaged into one or more vectors, e.g.,plasmid or viral vectors. In some embodiments, the vector, e.g., plasmidor viral vector is delivered to the tissue of interest by, for example,an intramuscular injection, while other times the delivery is viaintravenous, transdermal, intranasal, oral, mucosal, or other deliverymethods. Such delivery may be either via a single dose, or multipledoses. One skilled in the art understands that the actual dosage to bedelivered herein may vary greatly depending upon a variety of factors,such as the vector choice, the target cell, organism, or tissue, thegeneral condition of the subject to be treated, the degree oftransformation/modification sought, the administration route, theadministration mode, the type of transformation/modification sought,etc.

Such a dosage may further contain, for example, a carrier (water,saline, ethanol, glycerol, lactose, sucrose, calcium phosphate, gelatin,dextran, agar, pectin, peanut oil, sesame oil, etc.), a diluent, apharmaceutically-acceptable carrier (e.g., phosphate-buffered saline), apharmaceutically-acceptable excipient, and/or other compounds known inthe art. The dosage may further contain one or more pharmaceuticallyacceptable salts such as, for example, a mineral acid salt such as ahydrochloride, a hydrobromide, a phosphate, a sulfate, etc.; and thesalts of organic acids such as acetates, propionates, malonates,benzoates, etc. Additionally, auxiliary substances, such as wetting oremulsifying agents, pH buffering substances, gels or gelling materials,flavorings, colorants, microspheres, polymers, suspension agents, etc.may also be present herein. In addition, one or more other conventionalpharmaceutical ingredients, such as preservatives, humectants,suspending agents, surfactants, antioxidants, anticaking agents,fillers, chelating agents, coating agents, chemical stabilizers, etc.may also be present, especially if the dosage form is a reconstitutableform. Suitable exemplary ingredients include microcrystalline cellulose,carboxymethylcellulose sodium, polysorbate 80, phenylethyl alcohol,chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propylgallate, the parabens, ethyl vanillin, glycerin, phenol,parachlorophenol, gelatin, albumin and a combination thereof. A thoroughdiscussion of pharmaceutically acceptable excipients is available inREMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J. 1991) which isincorporated by reference herein.

In an embodiment herein the delivery is via an adenovirus, which may beat a single booster dose containing at least 1×10⁵ particles (alsoreferred to as particle units, pu) of adenoviral vector. In anembodiment herein, the dose preferably is at least about 1×10⁶ particles(for example, about 1×10⁶-1×10¹² particles), more preferably at leastabout 1×10⁷ particles, more preferably at least about 1×10⁸ particles(e.g., about 1×10⁸-1×10¹¹ particles or about 1×10⁸-1×10¹² particles),and most preferably at least about 1×10⁰ particles (e.g., about1×10⁹-1×10¹⁰ particles or about 1×10⁹-1×10¹² particles), or even atleast about 1×10¹⁰ particles (e.g., about 1×10¹⁰-1×10¹² particles) ofthe adenoviral vector. Alternatively, the dose comprises no more thanabout 1×10¹⁴ particles, preferably no more than about 1×10¹³ particles,even more preferably no more than about 1×10¹² particles, even morepreferably no more than about 1×10¹¹ particles, and most preferably nomore than about 1×10¹⁰ particles (e.g., no more than about 1×10⁹articles). Thus, the dose may contain a single dose of adenoviral vectorwith, for example, about 1×10⁶ particle units (pu), about 2×10⁶ pu,about 4×10⁶ pu, about 1×10⁷ pu, about 2×10′ pu, about 4×10⁷ pu, about1×10⁸ pu, about 2×10⁸ pu, about 4×10⁸ pu, about 1×10⁹ pu, about 2×10⁹pu, about 4×10⁹ pu, about 1×10¹⁰ pu, about 2×10¹⁰ pu, about 4×10¹⁰ pu,about 1×10¹¹ pu, about 2×10¹¹ pu, about 4×10¹¹ pu, about 1×10¹² pu,about 2×10¹² pu, or about 4×10¹² pu of adenoviral vector. See, forexample, the adenoviral vectors in U.S. Pat. No. 8,454,972 B2 to Nabel,et. al., granted on Jun. 4, 2013; incorporated by reference herein, andthe dosages at col 29, lines 36-58 thereof. In an embodiment herein, theadenovirus is delivered via multiple doses.

In an embodiment herein, the delivery is via an AAV. A therapeuticallyeffective dosage for in vivo delivery of the AAV to a human is believedto be in the range of from about 20 to about 50 ml of saline solutioncontaining from about 1×10¹⁰ to about 1×10¹⁰ functional AAV/ml solution.The dosage may be adjusted to balance the therapeutic benefit againstany side effects. In an embodiment herein, the AAV dose is generally inthe range of concentrations of from about 1×10⁵ to 1×10⁵⁰ genomes AAV,from about 1×10⁸ to 1×10²⁰ genomes AAV, from about 1×10¹⁰ to about1×10¹⁶ genomes, or about 1×10¹¹ to about 1×10¹⁶ genomes AAV. A humandosage may be about 1×10¹³ genomes AAV. Such concentrations may bedelivered in from about 0.001 ml to about 100 ml, about 0.05 to about 50ml, or about 10 to about 25 ml of a carrier solution. Other effectivedosages can be readily established by one of ordinary skill in the artthrough routine trials establishing dose response curves. See, forexample, U.S. Pat. No. 8,404,658 B2 to Hajjar, et al., granted on Mar.26, 2013, at col. 27, lines 45-60.

In an embodiment herein the delivery is via a plasmid. In such plasmidcompositions, the dosage should be a sufficient amount of plasmid toelicit a response. For instance, suitable quantities of plasmid DNA inplasmid compositions can be from about 0.1 to about 2 mg, or from about1 μg to about 10 μg per 70 kg individual. Plasmids of the invention willgenerally comprise (i) a promoter; (ii) a sequence encoding an nucleicacid-targeting CRISPR enzyme, operably linked to said promoter; (iii) aselectable marker; (iv) an origin of replication; and (v) atranscription terminator downstream of and operably linked to (ii). Theplasmid can also encode the RNA components of a CRISPR complex, but oneor more of these may instead be encoded on a different vector.

The doses herein are based on an average 70 kg individual. The frequencyof administration is within the ambit of the medical or veterinarypractitioner (e.g., physician, veterinarian), or scientist skilled inthe art. It is also noted that mice used in experiments are typicallyabout 20 g and from mice experiments one can scale up to a 70 kgindividual.

In some embodiments the RNA molecules of the invention are delivered inliposome or lipofectin formulations and the like and can be prepared bymethods well known to those skilled in the art. Such methods aredescribed, for example, in U.S. Pat. Nos. 5,593,972, 5,589,466, and5,580,859, which are herein incorporated by reference. Delivery systemsaimed specifically at the enhanced and improved delivery of siRNA intomammalian cells have been developed, (see, for example, Shen et al FEBSLet. 2003, 539:111-114; Xia et al., Nat. Biotech. 2002, 20:1006-1010;Reich et al., Mol. Vision. 2003, 9: 210-216; Sorensen et al., J. Mol.Biol. 2003, 327: 761-766; Lewis et al., Nat. Gen. 2002, 32: 107-108 andSimeoni et al., NAR 2003, 31, 11: 2717-2724) and may be applied to thepresent invention. siRNA has recently been successfully used forinhibition of gene expression in primates (see for example. Tolentino etal., Retina 24(4):660 which may also be applied to the presentinvention.

Indeed, RNA delivery is a useful method of in vivo delivery. It ispossible to deliver nucleic acid-targeting Cas protein and guide RNA(and, for instance, HR repair template) into cells using liposomes orparticles. Thus delivery of the nucleic acid-targeting Cas13b proteinand/or delivery of the guide RNAs or crRNAs of the invention may be inRNA form and via microvesicles, liposomes or particles. For example,Cas13b mRNA and guide RNA or crRNA can be packaged into liposomalparticles for delivery in vivo. Liposomal transfection reagents such aslipofectamine from Life Technologies and other reagents on the marketcan effectively deliver RNA molecules into the liver.

Means of delivery of RNA also preferred include delivery of RNA viananoparticles (Cho, S., Goldberg, M., Son, S., Xu, Q., Yang, F., Mei,Y., Bogatyrev, S., Langer, R. and Anderson, D., Lipid-like nanoparticlesfor small interfering RNA delivery to endothelial cells, AdvancedFunctional Materials, 19: 3112-3118, 2010) or exosomes (Schroeder, A.,Levins, C., Cortez, C., Langer, R., and Anderson, D., Lipid-basednanotherapeutics for siRNA delivery, Journal of Internal Medicine, 267:9-21, 2010, PMID: 20059641). Indeed, exosomes have been shown to beparticularly useful in delivery siRNA, a system with some parallels tothe RNA-targeting system. For instance, El-Andaloussi S, et al.(“Exosome-mediated delivery of siRNA in vitro and in vivo.” Nat Protoc.2012 Dec.; 7(12):2112-26. doi: 10.1038/nprot.2012.131. Epub 2012 Nov.15.) describe how exosomes are promising tools for drug delivery acrossdifferent biological barriers and can be harnessed for delivery of siRNAin vitro and in vivo. Their approach is to generate targeted exosomesthrough transfection of an expression vector, comprising an exosomalprotein fused with a peptide ligand. The exosomes are then purify andcharacterized from transfected cell supernatant, then RNA is loaded intothe exosomes. Delivery or administration according to the invention canbe performed with exosomes, in particular but not limited to the brain.Vitamin E (α-tocopherol) may be conjugated with nucleic acid-targetingCas protein and delivered to the brain along with high densitylipoprotein (HDL), for example in a similar manner as was done by Uno etal. (HUMAN GENE THERAPY 22:711-719 (June 2011)) for deliveringshort-interfering RNA (siRNA) to the brain. Mice were infused viaOsmotic minipumps (model 1007D; Alzet, Cupertino, CA) filled withphosphate-buffered saline (PBS) or free TocsiBACE or Toc-siBACE/HDL andconnected with Brain Infusion Kit 3 (Alzet). A brain-infusion cannulawas placed about 0.5 mm posterior to the bregma at midline for infusioninto the dorsal third ventricle. Uno et al. found that as little as 3nmol of Toc-siRNA with HDL could induce a target reduction in comparabledegree by the same ICV infusion method. A similar dosage of nucleicacid-targeting effector protein conjugated to α-tocopherol andco-administered with HDL targeted to the brain may be contemplated forhumans in the present invention, for example, about 3 nmol to about 3μmol of nucleic acid-targeting effector protein targeted to the brainmay be contemplated. Zou et al. ((HUMAN GENE THERAPY 22:465-475 (April2011)) describes a method of lentiviral-mediated delivery ofshort-hairpin RNAs targeting PKCγ for in vivo gene silencing in thespinal cord of rats. Zou et al. administered about 10 μl of arecombinant lentivirus having a titer of 1×10⁹ transducing units (TU)/mlby an intrathecal catheter. A similar dosage of nucleic acid-targetingeffector protein expressed in a lentiviral vector targeted to the brainmay be contemplated for humans in the present invention, for example,about 10-50 ml of nucleic acid-targeting effector protein targeted tothe brain in a lentivirus having a titer of 1×10⁹ transducing units(TU)/ml may be contemplated.

In terms of local delivery to the brain, this can be achieved in variousways. For instance, material can be delivered intrastriatally e.g., byinjection. Injection can be performed stereotactically via a craniotomy.

Packaging and Promoters Generally

Ways to package RNA-targeting effector protein (Cas13b proteins) codingnucleic acid molecules, e.g., DNA, into vectors, e.g., viral vectors, tomediate genome modification in vivo include:

-   -   Single virus vector:        -   Vector containing two or more expression cassettes:        -   Promoter-nucleic acid-targeting effector protein coding            nucleic acid molecule-terminator        -   Promoter-guide RNAI-terminator        -   Promoter-guide RNA (N)-terminator (up to size limit of            vector)    -   Double virus vector:        -   Vector 1 containing one expression cassette for driving the            expression of RNA-targeting effector protein (Cas13b)        -   Promoter-RNA-targeting effector (Cas13b) protein coding            nucleic acid molecule-terminator        -   Vector 2 containing one more expression cassettes for            driving the expression of one or more guide RNAs or crRNAs        -   Promoter-guide RNA1 or crRNA1-terminator        -   Promoter-guide RNA1 (N) or crRNA1 (N)-terminator (up to size            limit of vector).

The promoter used to drive RNA-targeting effector protein coding nucleicacid molecule expression can include AAV ITR can serve as a promoter:this is advantageous for eliminating the need for an additional promoterelement (which can take up space in the vector). The additional spacefreed up can be used to drive the expression of additional elements(gRNA, etc.). Also, ITR activity is relatively weaker, so can be used toreduce potential toxicity due to over expression of nucleicacid-targeting effector protein. For ubiquitous expression, can usepromoters: CMV, CAG, CBh, PGK, SV40, Ferritin heavy or light chains,etc. For brain or other CNS expression, can use promoters: SynapsinI forall neurons, CaMKIIalpha for excitatory neurons, GAD67 or GAD65 or VGATfor GABAergic neurons, etc. For liver expression, can use Albuminpromoter. For lung expression, can use SP-B. For endothelial cells, canuse ICAM. For hematopoietic cells can use IFNbeta or CD45. ForOsteoblasts can use OG-2. The promoter used to drive guide RNA caninclude: Pol III promoters such as U6 or H1; Pol II promoter andintronic cassettes to express guide RNA or crRNA.

Adeno Associated Virus (AAV)

Cas13b and one or more guide RNA or crRNA can be delivered using adenoassociated virus (AAV), lentivirus, adenovirus or other plasmid or viralvector types, in particular, using formulations and doses from, forexample, U.S. Pat. No. 8,454,972 (formulations, doses for adenovirus),U.S. Pat. No. 8,404,658 (formulations, doses for AAV) and U.S. Pat. No.5,846,946 (formulations, doses for DNA plasmids) and from clinicaltrials and publications regarding the clinical trials involvinglentivirus, AAV and adenovirus. For examples, for AAV, the route ofadministration, formulation and dose can be as in U.S. Pat. No.8,454,972 and as in clinical trials involving AAV. For Adenovirus, theroute of administration, formulation and dose can be as in U.S. Pat. No.8,404,658 and as in clinical trials involving adenovirus. For plasmiddelivery, the route of administration, formulation and dose can be as inU.S. Pat. No. 5,846,946 and as in clinical studies involving plasmids.Doses may be based on or extrapolated to an average 70 kg individual(e.g., a male adult human), and can be adjusted for patients, subjects,mammals of different weight and species. Frequency of administration iswithin the ambit of the medical or veterinary practitioner (e.g.,physician, veterinarian), depending on usual factors including the age,sex, general health, other conditions of the patient or subject and theparticular condition or symptoms being addressed. The viral vectors canbe injected into the tissue of interest. For cell-type specific genomemodification, the expression of RNA-targeting effector protein (Cas13beffector protein) can be driven by a cell-type specific promoter. Forexample, liver-specific expression might use the Albumin promoter andneuron-specific expression (e.g., for targeting CNS disorders) might usethe Synapsin I promoter. In terms of in vivo delivery, AAV isadvantageous over other viral vectors for a couple of reasons: Lowtoxicity (this may be due to the purification method not requiring ultracentrifugation of cell particles that can activate the immune response)and Low probability of causing insertional mutagenesis because itdoesn't integrate into the host genome.

AAV has a packaging limit of 4.5 or 4.75 Kb. This means that theRNA-targeting effector protein (Cas13b effector protein) coding sequenceas well as a promoter and transcription terminator have to be all fitinto the same viral vector. As to AAV, the AAV can be AAV1, AAV2, AAV5or any combination thereof. One can select the AAV of the AAV withregard to the cells to be targeted; e.g., one can select AAV serotypes1, 2, 5 or a hybrid capsid AAV1, AAV2, AAV5 or any combination thereoffor targeting brain or neuronal cells; and one can select AAV4 fortargeting cardiac tissue. AAV8 is useful for delivery to the liver. Theherein promoters and vectors are preferred individually. A tabulation ofcertain AAV serotypes as to these cells (see Grimm, D. et al, J. Virol.82: 5887-5911 (2008)) is as follows:

Cell Line AAV-1 AAV-2 AAV-3 AAV-4 AAV-5 AAV-6 AAV-8 AAV-9 Huh-7 13 1002.5 0.0 0.1 10 0.7 0.0 HEK293 25 100 2.5 0.1 0.1 5 0.7 0.1 HeLa 3 1002.0 0.1 6.7 1 0.2 0.1 HepG2 3 100 16.7 0.3 1.7 5 0.3 ND Hep1A 20 100 0.21.0 0.1 1 0.2 0.0 911 17 100 11 0.2 0.1 17 0.1 ND CHO 100 100 14 1.4 33350 10 1.0 COS 33 100 33 3.3 5.0 14 2.0 0.5 MeWo 10 100 20 0.3 6.7 10 1.00.2 NIH3T3 10 100 2.9 2.9 0.3 10 0.3 ND A549 14 100 20 ND 0.5 10 0.5 0.1HT1180 20 100 10 0.1 0.3 33 0.5 0.1 Monocytes 1111 100 ND ND 125 1429 NDND Immature DC 2500 100 ND ND 222 2857 ND ND Mature DC 2222 100 ND ND333 3333 ND ND

Lentivirus

Lentiviruses are complex retroviruses that have the ability to infectand express their genes in both mitotic and post-mitotic cells. The mostcommonly known lentivirus is the human immunodeficiency virus (HIV),which uses the envelope glycoproteins of other viruses to target a broadrange of cell types. Lentiviruses may be prepared as follows. Aftercloning pCasES10 (which contains a lentiviral transfer plasmidbackbone), HEK293FT at low passage (p=5) were seeded in a T-75 flask to50% confluence the day before transfection in DMEM with 10% fetal bovineserum and without antibiotics. After 20 hours, media was changed toOptiMEM (serum-free) media and transfection was done 4 hours later.Cells were transfected with 10 μg of lentiviral transfer plasmid(pCasES10) and the following packaging plasmids: 5 of pMD2.G (VSV-gpseudotype), and 7.5 ug of psPAX2 (gag/pol/rev/tat). Transfection wasdone in 4 mL OptiMEM with a cationic lipid delivery agent (50 uLLipofectamine 2000 and 100 ul Plus reagent). After 6 hours, the mediawas changed to antibiotic-free DMEM with 10% fetal bovine serum. Thesemethods use serum during cell culture, but serum-free methods arepreferred.

Lentivirus may be purified as follows. Viral supernatants were harvestedafter 48 hours. Supernatants were first cleared of debris and filteredthrough a 0.45 um low protein binding (PVDF) filter. They were then spunin a ultracentrifuge for 2 hours at 24,000 rpm. Viral pellets wereresuspended in 50 ul of DMEM overnight at 4C. They were then aliquotedand immediately frozen at −80° C.

In another embodiment, minimal non-primate lentiviral vectors based onthe equine infectious anemia virus (EIAV) are also contemplated,especially for ocular gene therapy (see, e.g., Balagaan, J Gene Med2006; 8: 275-285). In another embodiment, RetinoStat®, an equineinfectious anemia virus-based lentiviral gene therapy vector thatexpresses angiostatic proteins endostatin and angiostatin that isdelivered via a subretinal injection for the treatment of the web formof age-related macular degeneration is also contemplated (see, e.g.,Binley et al., HUMAN GENE THERAPY 23:980-991 (September 2012)) and thisvector may be modified for the nucleic acid-targeting system of thepresent invention.

In another embodiment, self-inactivating lentiviral vectors with ansiRNA targeting a common exon shared by HIV tat/rev, anucleolar-localizing TAR decoy, and an anti-CCR5-specific hammerheadribozyme (see, e.g., DiGiusto et al. (2010) Sci Transl Med 2:36ra43) maybe used/and or adapted to the nucleic acid-targeting system of thepresent invention. A minimum of 2.5×10⁶ CD34+ cells per kilogram patientweight may be collected and prestimulated for 16 to 20 hours in X-VIVO15 medium (Lonza) containing 2 μmol/L-glutamine, stem cell factor (100ng/ml), Flt-3 ligand (Flt-3L) (100 ng/ml), and thrombopoietin (10 ng/ml)(CellGenix) at a density of 2×10⁶ cells/ml. Prestimulated cells may betransduced with lentiviral at a multiplicity of infection of 5 for 16 to24 hours in 75-cm² tissue culture flasks coated with fibronectin (25mg/cm²) (RetroNectin, Takara Bio Inc.).

Lentiviral vectors have been disclosed as in the treatment forParkinson's Disease, see, e.g., US Patent Publication No. 20120295960and U.S. Pat. Nos. 7,303,910 and 7,351,585. Lentiviral vectors have alsobeen disclosed for the treatment of ocular diseases, see e.g., US PatentPublication Nos. 20060281180, 20090007284, US20110117189; US20090017543;US20070054961, US20100317109. Lentiviral vectors have also beendisclosed for delivery to the brain, see, e.g., US Patent PublicationNos. US20110293571; US20110293571, US20040013648, US20070025970,US20090111106 and U.S. Pat. No. 7,259,015.

RNA Delivery

RNA delivery: The nucleic acid-targeting Cas13b protein, and/or guideRNA, can also be delivered in the form of RNA. mRNA can be synthesizedusing a PCR cassette containing the following elements:T7_promoter-kozak sequence (GCCACC)-effector protein-3′ UTR from betaglobin-polyA tail (a string of 120 or more adenines). The cassette canbe used for transcription by T7 polymerase. Guide RNAs or crRNAs canalso be transcribed using in vitro transcription from a cassettecontaining T7 promoter-GG-guide RNA or crRNA sequence.

Particle Delivery Systems and/or Formulations:

Several types of particle delivery systems and/or formulations are knownto be useful in a diverse spectrum of biomedical applications. Ingeneral, a particle is defined as a small object that behaves as a wholeunit with respect to its transport and properties. Particles are furtherclassified according to diameter. Coarse particles cover a range between2,500 and 10,000 nanometers. Fine particles are sized between 100 and2,500 nanometers. Ultrafine particles, or nanoparticles, are generallybetween 1 and 100 nanometers in size. The basis of the 100-nm limit isthe fact that novel properties that differentiate particles from thebulk material typically develop at a critical length scale of under 100nm.

As used herein, a particle delivery system/formulation is defined as anybiological delivery system/formulation which includes a particle inaccordance with the present invention. A particle in accordance with thepresent invention is any entity having a greatest dimension (e.g.diameter) of less than 100 microns (μm). In some embodiments, inventiveparticles have a greatest dimension of less than 10 μm. In someembodiments, inventive particles have a greatest dimension of less than2000 nanometers (nm). In some embodiments, inventive particles have agreatest dimension of less than 1000 nanometers (nm). In someembodiments, inventive particles have a greatest dimension of less than900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, or 100nm. Typically, inventive particles have a greatest dimension (e.g.,diameter) of 500 nm or less. In some embodiments, inventive particleshave a greatest dimension (e.g., diameter) of 250 nm or less. In someembodiments, inventive particles have a greatest dimension (e.g.,diameter) of 200 nm or less. In some embodiments, inventive particleshave a greatest dimension (e.g., diameter) of 150 nm or less. In someembodiments, inventive particles have a greatest dimension (e.g.,diameter) of 100 nm or less. Smaller particles, e.g., having a greatestdimension of 50 nm or less are used in some embodiments of theinvention. In some embodiments, inventive particles have a greatestdimension ranging between 25 nm and 200 nm.

Particle characterization (including e.g., characterizing morphology,dimension, etc.) is done using a variety of different techniques. Commontechniques are electron microscopy (TEM, SEM), atomic force microscopy(AFM), dynamic light scattering (DLS), X-ray photoelectron spectroscopy(XPS), powder X-ray diffraction (XRD), Fourier transform infraredspectroscopy (FTIR), matrix-assisted laser desorption/ionizationtime-of-flight mass spectrometry(MALDI-TOF), ultraviolet-visiblespectroscopy, dual polarisation interferometry and nuclear magneticresonance (NMR). Characterization (dimension measurements) may be madeas to native particles (i.e., preloading) or after loading of the cargo(herein cargo refers to e.g., one or more components of CRISPR-Cas13bsystem e.g., Cas13b enzyme or mRNA or guide RNA, or any combinationthereof, and may include additional carriers and/or excipients) toprovide particles of an optimal size for delivery for any in vitro, exvivo and/or in vivo application of the present invention. In certainpreferred embodiments, particle dimension (e.g., diameter)characterization is based on measurements using dynamic laser scattering(DLS). Mention is made of U.S. Pat. Nos. 8,709,843; 6,007,845;5,855,913; U.S. Pat. Nos. 5,985,309; 5,543,158; and the publication byJames E. Dahlman and Carmen Barnes et al. Nature Nanotechnology (2014)published online 11 May 2014, doi:10.1038/nnano.2014.84, concerningparticles, methods of making and using them and measurements thereof.See also Dahlman et al. “Orthogonal gene control with a catalyticallyactive Cas9 nuclease,” Nature Biotechnology 33, 1159-1161 (November,2015).

Particles delivery systems within the scope of the present invention maybe provided in any form, including but not limited to, solid,semi-solid, emulsion, or colloidal particles. As such any of thedelivery systems described herein, including but not limited to, e.g.,lipid-based systems, liposomes, micelles, microvesicles, exosomes, orgene gun may be provided as particle delivery systems within the scopeof the present invention.

Particles

Cas13b mRNA and guide RNA or crRNA may be delivered simultaneously usingparticles or lipid envelopes; for instance, CRISPR enzyme and RNA of theinvention, e.g., as a complex, can be delivered via a particle as inDahlman et al., WO 2015/089419 A2 and documents cited therein, such as7C1 (see, e.g., James E. Dahlman and Carmen Barnes et al. NatureNanotechnology (2014) published online 11 May 2014,doi:10.1038/nnano.2014.84), e.g., delivery particle comprising lipid orlipidoid and hydrophilic polymer, e.g., cationic lipid and hydrophilicpolymer, for instance wherein the the cationic lipid comprises1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or1,2-ditetradecanoyl-sn-glycero-3-phosphocholine (DMPC) and/or whereinthe hydrophilic polymer comprises ethylene glycol or polyethylene glycol(PEG); and/or wherein the particle further comprises cholesterol (e.g.,particle from formulation 1=DOTAP 100, DMPC 0, PEG 0, Cholesterol 0;formulation number 2=DOTAP 90, DMPC 0, PEG 10, Cholesterol 0;formulation number 3=DOTAP 90, DMPC 0, PEG 5, Cholesterol 5), whereinparticles are formed using an efficient, multistep process whereinfirst, effector protein and RNA are mixed together, e.g., at a 1:1 molarratio, e.g., at room temperature, e.g., for 30 minutes, e.g., insterile, nuclease free 1X PBS; and separately, DOTAP, DMPC, PEG, andcholesterol as applicable for the formulation are dissolved in alcohol,e.g., 100% ethanol; and, the two solutions are mixed together to formparticles containing the complexes). Cas13b effector protein mRNA andguide RNA may be delivered simultaneously using particles or lipidenvelopes. This Dahlman et al technology can be applied in the instantinvention. An epoxide-modified lipid-polymer may be utilized to deliverthe nucleic acid-targeting system of the present invention to pulmonary,cardiovascular or renal cells, however, one of skill in the art mayadapt the system to deliver to other target organs. Dosage ranging fromabout 0.05 to about 0.6 mg/kg are envisioned. Dosages over several daysor weeks are also envisioned, with a total dosage of about 2 mg/kg. Forexample, Su X, Fricke J, Kavanagh D G, Irvine D J (“In vitro and in vivomRNA delivery using lipid-enveloped pH-responsive polymer nanoparticles”Mol Pharm. 2011 Jun. 6; 8(3):774-87. doi: 10.1021/mp100390w. Epub 2011Apr. 1) describes biodegradable core-shell structured particles with apoly(β-amino ester) (PBAE) core enveloped by a phospholipid bilayershell. These were developed for in vivo mRNA delivery. The pH-responsivePBAE component was chosen to promote endosome disruption, while thelipid surface layer was selected to minimize toxicity of the polycationcore. Such are, therefore, preferred for delivering RNA of the presentinvention.

In one embodiment, particles based on self-assembling bioadhesivepolymers are contemplated, which may be applied to oral delivery ofpeptides, intravenous delivery of peptides and nasal delivery ofpeptides, all to the brain. Other embodiments, such as oral absorptionand ocular delivery of hydrophobic drugs are also contemplated. Themolecular envelope technology involves an engineered polymer envelopewhich is protected and delivered to the site of the disease (see, e.g.,Mazza, M. et al. ACSNano, 2013. 7(2): 1016-1026; Siew, A., et al. MolPharm, 2012. 9(1):14-28; Lalatsa, A., et al. J Contr Rel, 2012.161(2):523-36; Lalatsa, A., et al., Mol Pharm, 2012. 9(6):1665-80;Lalatsa, A., et al. Mol Pharm, 2012. 9(6):1764-74; Garrett, N. L., etal. J Biophotonics, 2012. 5(5-6):458-68; Garrett, N. L., et al. J RamanSpect, 2012. 43(5):681-688; Ahmad, S., et al. J Royal Soc Interface2010. 7:S423-33; Uchegbu, I. F. Expert Opin Drug Deliv, 2006.3(5):629-40; Qu, X., et al. Biomacromolecules, 2006. 7(12):3452-9 andUchegbu, I. F., et al. Int J Pharm, 2001.224:185-199). Doses of about 5mg/kg are contemplated, with single or multiple doses, depending on thetarget tissue.

Regarding particles, see, also Alabi et al., Proc Natl Acad Sci USA.2013 Aug. 6; 110(32):12881-6; Zhang et al., Adv Mater. 2013 Sep. 6;25(33):4641-5; Jiang et al., Nano Lett. 2013 Mar. 13; 13(3):1059-64;Karagiannis et al., ACS Nano. 2012 Oct. 23; 6(10):8484-7; Whitehead etal., ACS Nano. 2012 Aug. 28; 6(8):6922-9 and Lee et al., NatNanotechnol. 2012 Jun. 3; 7(6):389 93.

US patent application 20110293703 relates to lipidoid compounds are alsoparticularly useful in the administration of polynucleotides, which maybe applied to deliver the nucleic acid-targeting system of the presentinvention. In one aspect, the aminoalcohol lipidoid compounds arecombined with an agent to be delivered to a cell or a subject to formmicroparticles, nanoparticles, liposomes, or micelles. The agent to bedelivered by the particles, liposomes, or micelles may be in the form ofa gas, liquid, or solid, and the agent may be a polynucleotide, protein,peptide, or small molecule. The aminoalcohol lipidoid compounds may becombined with other aminoalcohol lipidoid compounds, polymers (syntheticor natural), surfactants, cholesterol, carbohydrates, proteins, lipids,etc. to form the particles. These particles may then optionally becombined with a pharmaceutical excipient to form a pharmaceuticalcomposition. US Patent Publication No. 20110293703 also provides methodsof preparing the aminoalcohol lipidoid compounds. One or moreequivalents of an amine are allowed to react with one or moreequivalents of an epoxide-terminated compound under suitable conditionsto form an aminoalcohol lipidoid compound of the present invention. Incertain embodiments, all the amino groups of the amine are fully reactedwith the epoxide-terminated compound to form tertiary amines. In otherembodiments, all the amino groups of the amine are not fully reactedwith the epoxide-terminated compound to form tertiary amines therebyresulting in primary or secondary amines in the aminoalcohol lipidoidcompound. These primary or secondary amines are left as is or may bereacted with another electrophile such as a different epoxide-terminatedcompound. As will be appreciated by one skilled in the art, reacting anamine with less than excess of epoxide-terminated compound will resultin a plurality of different aminoalcohol lipidoid compounds with variousnumbers of tails. Certain amines may be fully functionalized with twoepoxide-derived compound tails while other molecules will not becompletely functionalized with epoxide-derived compound tails. Forexample, a diamine or polyamine may include one, two, three, or fourepoxide-derived compound tails off the various amino moieties of themolecule resulting in primary, secondary, and tertiary amines. Incertain embodiments, all the amino groups are not fully functionalized.In certain embodiments, two of the same types of epoxide-terminatedcompounds are used. In other embodiments, two or more differentepoxide-terminated compounds are used. The synthesis of the aminoalcohollipidoid compounds is performed with or without solvent, and thesynthesis may be performed at higher temperatures ranging from 30-100°C., preferably at approximately 50-90° C. The prepared aminoalcohollipidoid compounds may be optionally purified. For example, the mixtureof aminoalcohol lipidoid compounds may be purified to yield anaminoalcohol lipidoid compound with a particular number ofepoxide-derived compound tails. Or the mixture may be purified to yielda particular stereo- or regioisomer. The aminoalcohol lipidoid compoundsmay also be alkylated using an alkyl halide (e.g., methyl iodide) orother alkylating agent, and/or they may be acylated.

US Patent Publication No. 20110293703 also provides libraries ofaminoalcohol lipidoid compounds prepared by the inventive methods. Theseaminoalcohol lipidoid compounds may be prepared and/or screened usinghigh-throughput techniques involving liquid handlers, robots, microtiterplates, computers, etc. In certain embodiments, the aminoalcohollipidoid compounds are screened for their ability to transfectpolynucleotides or other agents (e.g., proteins, peptides, smallmolecules) into the cell. US Patent Publication No. 20130302401 relatesto a class of poly(beta-amino alcohols) (PBAAs) has been prepared usingcombinatorial polymerization. The inventive PBAAs may be used inbiotechnology and biomedical applications as coatings (such as coatingsof films or multilayer films for medical devices or implants),additives, materials, excipients, non-biofouling agents, micropatterningagents, and cellular encapsulation agents. When used as surfacecoatings, these PBAAs elicited different levels of inflammation, both invitro and in vivo, depending on their chemical structures. The largechemical diversity of this class of materials allowed us to identifypolymer coatings that inhibit macrophage activation in vitro.Furthermore, these coatings reduce the recruitment of inflammatorycells, and reduce fibrosis, following the subcutaneous implantation ofcarboxylated polystyrene microparticles. These polymers may be used toform polyelectrolyte complex capsules for cell encapsulation. Theinvention may also have many other biological applications such asantimicrobial coatings, DNA or siRNA delivery, and stem cell tissueengineering. The teachings of US Patent Publication No. 20130302401 maybe applied to the nucleic acid-targeting system of the presentinvention.

In another embodiment, lipid nanoparticles (LNPs) are contemplated. Anantitransthyretin small interfering RNA has been encapsulated in lipidnanoparticles and delivered to humans (see, e.g., Coelho et al., N EnglJ Med 2013; 369:819-29), and such a system may be adapted and applied tothe nucleic acid-targeting system of the present invention. Doses ofabout 0.01 to about 1 mg per kg of body weight administeredintravenously are contemplated. Medications to reduce the risk ofinfusion-related reactions are contemplated, such as dexamethasone,acetampinophen, diphenhydramine or cetirizine, and ranitidine arecontemplated. Multiple doses of about 0.3 mg per kilogram every 4 weeksfor five doses are also contemplated. LNPs have been shown to be highlyeffective in delivering siRNAs to the liver (see, e.g., Tabernero etal., Cancer Discovery, April 2013, Vol. 3, No. 4, pages 363-470) and aretherefore contemplated for delivering RNA encoding nucleicacid-targeting effector protein to the liver. A dosage of about fourdoses of 6 mg/kg of the LNP every two weeks may be contemplated.Tabernero et al. demonstrated that tumor regression was observed afterthe first 2 cycles of LNPs dosed at 0.7 mg/kg, and by the end of 6cycles the patient had achieved a partial response with completeregression of the lymph node metastasis and substantial shrinkage of theliver tumors. A complete response was obtained after 40 doses in thispatient, who has remained in remission and completed treatment afterreceiving doses over 26 months. Two patients with RCC and extrahepaticsites of disease including kidney, lung, and lymph nodes that wereprogressing following prior therapy with VEGF pathway inhibitors hadstable disease at all sites for approximately 8 to 12 months, and apatient with PNET and liver metastases continued on the extension studyfor 18 months (36 doses) with stable disease. However, the charge of theLNP must be taken into consideration. As cationic lipids combined withnegatively charged lipids to induce nonbilayer structures thatfacilitate intracellular delivery. Because charged LNPs are rapidlycleared from circulation following intravenous injection, ionizablecationic lipids with pKa values below 7 were developed (see, e.g., Rosinet al, Molecular Therapy, vol. 19, no. 12, pages 1286-2200, December2011). Negatively charged polymers such as RNA may be loaded into LNPsat low pH values (e.g., pH 4) where the ionizable lipids display apositive charge. However, at physiological pH values, the LNPs exhibit alow surface charge compatible with longer circulation times. Fourspecies of ionizable cationic lipids have been focused upon, namely1,2-dilinoleoyl-3-dimethylammonium-propane (DLinDAP),1,2-dilinoleyloxy-3-N,N-dimethylaminopropane (DLinDMA),1,2-dilinoleyloxy-keto-N,N-dimethyl-3-aminopropane (DLinKDMA), and1,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2-DMA).It has been shown that LNP siRNA systems containing these lipids exhibitremarkably different gene silencing properties in hepatocytes in vivo,with potencies varying according to the seriesDLinKC2-DMA>DLinKDMA>DLinDMA>>DLinDAP employing a Factor VII genesilencing model (see, e.g., Rosin et al, Molecular Therapy, vol. 19, no.12, pages 1286-2200, December 2011). A dosage of 1 μg/ml of LNP orCRISPR-Cas RNA in or associated with the LNP may be contemplated,especially for a formulation containing DLinKC2-DMA.

Preparation of LNPs and CRISPR-Cas13b encapsulation may be used/and oradapted from Rosin et al, Molecular Therapy, vol. 19, no. 12, pages1286-2200, December 2011). The cationic lipids1,2-dilinoleoyl-3-dimethylammonium-propane (DLinDAP),1,2-dilinoleyloxy-3-N,N-dimethylaminopropane (DLinDMA),1,2-dilinoleyloxyketo-N,N-dimethyl-3-aminopropane (DLinK-DMA),1,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2-DMA),(3-o-[2″-(methoxypolyethyleneglycol 2000)succinoyl]-1,2-dimyristoyl-sn-glycol (PEG-S-DMG), andR-3-[(w-methoxy-poly(ethylene glycol)2000)carbamoyl]-1,2-dimyristyloxlpropyl-3-amine (PEG-C-DOMG) may be providedby Tekmira Pharmaceuticals (Vancouver, Canada) or synthesized.Cholesterol may be purchased from Sigma (St Louis, MO). The specificnucleic acid-targeting complex (CRISPR-Cas) RNA may be encapsulated inLNPs containing DLinDAP, DLinDMA, DLinK-DMA, and DLinKC2-DMA (cationiclipid:DSPC:CHOL: PEGS-DMG or PEG-C-DOMG at 40:10:40:10 molar ratios).When required, 0.2% SP-DiOC18 (Invitrogen, Burlington, Canada) may beincorporated to assess cellular uptake, intracellular delivery, andbiodistribution. Encapsulation may be performed by dissolving lipidmixtures comprised of cationic lipid:DSPC:cholesterol:PEG-c-DOMG(40:10:40:10 molar ratio) in ethanol to a final lipid concentration of10 mmol/l. This ethanol solution of lipid may be added drop-wise to 50mmol/l citrate, pH 4.0 to form multilamellar vesicles to produce a finalconcentration of 30% ethanol vol/vol. Large unilamellar vesicles may beformed following extrusion of multilamellar vesicles through two stacked80 nm Nuclepore polycarbonate filters using the Extruder (NorthernLipids, Vancouver, Canada). Encapsulation may be achieved by adding RNAdissolved at 2 mg/ml in 50 mmol/l citrate, pH 4.0 containing 30% ethanolvol/vol drop-wise to extruded preformed large unilamellar vesicles andincubation at 31° C. for 30 minutes with constant mixing to a finalRNA/lipid weight ratio of 0.06/1 wt/wt. Removal of ethanol andneutralization of formulation buffer were performed by dialysis againstphosphate-buffered saline (PBS), pH 7.4 for 16 hours using Spectra/Por 2regenerated cellulose dialysis membranes. Particle size distribution maybe determined by dynamic light scattering using a NICOMP 370 particlesizer, the vesicle/intensity modes, and Gaussian fitting (NicompParticle Sizing, Santa Barbara, CA). The particle size for all three LNPsystems may be ˜70 nm in diameter. RNA encapsulation efficiency may bedetermined by removal of free RNA using VivaPureD MiniH columns(Sartorius Stedim Biotech) from samples collected before and afterdialysis. The encapsulated RNA may be extracted from the elutedparticles and quantified at 260 nm. RNA to lipid ratio was determined bymeasurement of cholesterol content in vesicles using the Cholesterol Eenzymatic assay from Wako Chemicals USA (Richmond, VA). In conjunctionwith the herein discussion of LNPs and PEG lipids, PEGylated liposomesor LNPs are likewise suitable for delivery of a nucleic acid-targetingsystem or components thereof. Preparation of large LNPs may be used/andor adapted from Rosin et al, Molecular Therapy, vol. 19, no. 12, pages1286-2200, December 2011. A lipid premix solution (20.4 mg/ml totallipid concentration) may be prepared in ethanol containing DLinKC2-DMA,DSPC, and cholesterol at 50:10:38.5 molar ratios. Sodium acetate may beadded to the lipid premix at a molar ratio of 0.75:1 (sodiumacetate:DLinKC2-DMA). The lipids may be subsequently hydrated bycombining the mixture with 1.85 volumes of citrate buffer (10 mmol/l, pH3.0) with vigorous stirring, resulting in spontaneous liposome formationin aqueous buffer containing 35% ethanol. The liposome solution may beincubated at 37° C. to allow for time-dependent increase in particlesize. Aliquots may be removed at various times during incubation toinvestigate changes in liposome size by dynamic light scattering(Zetasizer Nano ZS, Malvern Instruments, Worcestershire, UK). Once thedesired particle size is achieved, an aqueous PEG lipid solution(stock=10 mg/ml PEG-DMG in 35% (vol/vol) ethanol) may be added to theliposome mixture to yield a final PEG molar concentration of 3.5% oftotal lipid. Upon addition of PEG-lipids, the liposomes should theirsize, effectively quenching further growth. RNA may then be added to theempty liposomes at a RNA to total lipid ratio of approximately 1:10(wt:wt), followed by incubation for 30 minutes at 37° C. to form loadedLNPs. The mixture may be subsequently dialyzed overnight in PBS andfiltered with a 0.45-μm syringe filter.

Spherical Nucleic Acid (SNA™) constructs and other particles(particularly gold particles) are also contemplated as a means todelivery nucleic acid-targeting system to intended targets. Significantdata show that AuraSense Therapeutics' Spherical Nucleic Acid (SNA™)constructs, based upon nucleic acid-functionalized gold particles, areuseful.

Literature that may be employed in conjunction with herein teachingsinclude: Cutler et al., J. Am. Chem. Soc. 2011 133:9254-9257, Hao etal., Small. 2011 7:3158-3162, Zhang et al., ACS Nano. 2011 5:6962-6970,Cutler et al., J. Am. Chem. Soc. 2012 134:1376-1391, Young et al., NanoLett. 2012 12:3867-71, Zheng et al., Proc. Natl. Acad. Sci. USA. 2012109:11975-80, Mirkin, Nanomedicine 2012 7:635-638 Zhang et al., J. Am.Chem. Soc. 2012 134:16488-1691, Weintraub, Nature 2013 495:S14-S16, Choiet al., Proc. Natl. Acad. Sci. USA. 2013 110(19):7625 7630, Jensen etal., Sci. Transl. Med. 5, 209ra152 (2013) and Mirkin, et al., Small,10:186-192.

Self-assembling particles with RNA may be constructed withpolyethyleneimine (PEI) that is PEGylated with an Arg-Gly-Asp (RGD)peptide ligand attached at the distal end of the polyethylene glycol(PEG). This system has been used, for example, as a means to targettumor neovasculature expressing integrins and deliver siRNA inhibitingvascular endothelial growth factor receptor-2 (VEGF R2) expression andthereby achieve tumor angiogenesis (see, e.g., Schiffelers et al.,Nucleic Acids Research, 2004, Vol. 32, No. 19). Nanoplexes may beprepared by mixing equal volumes of aqueous solutions of cationicpolymer and nucleic acid to give a net molar excess of ionizablenitrogen (polymer) to phosphate (nucleic acid) over the range of 2 to 6.The electrostatic interactions between cationic polymers and nucleicacid resulted in the formation of polyplexes with average particle sizedistribution of about 100 nm, hence referred to here as nanoplexes. Adosage of about 100 to 200 mg of nucleic acid-targeting complex RNA isenvisioned for delivery in the self-assembling particles of Schiffelerset al.

The nanoplexes of Bartlett et al. (PNAS, Sep. 25, 2007, vol. 104, no.39) may also be applied to the present invention. The nanoplexes ofBartlett et al. are prepared by mixing equal volumes of aqueoussolutions of cationic polymer and nucleic acid to give a net molarexcess of ionizable nitrogen (polymer) to phosphate (nucleic acid) overthe range of 2 to 6. The electrostatic interactions between cationicpolymers and nucleic acid resulted in the formation of polyplexes withaverage particle size distribution of about 100 nm, hence referred tohere as nanoplexes. The DOTA-siRNA of Bartlett et al. was synthesized asfollows: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acidmono(N-hydroxysuccinimide ester) (DOTA-NHSester) was ordered fromMacrocyclics (Dallas, TX). The amine modified RNA sense strand with a100-fold molar excess of DOTA-NHS-ester in carbonate buffer (pH 9) wasadded to a microcentrifuge tube. The contents were reacted by stirringfor 4 h at room temperature. The DOTA-RNA sense conjugate wasethanol-precipitated, resuspended in water, and annealed to theunmodified antisense strand to yield DOTA-siRNA. All liquids werepretreated with Chelex-100 (Bio-Rad, Hercules, CA) to remove trace metalcontaminants. Tf-targeted and nontargeted siRNA particles may be formedby using cyclodextrin-containing polycations. Typically, particles wereformed in water at a charge ratio of 3 (+/−) and an siRNA concentrationof 0.5 g/liter. One percent of the adamantane-PEG molecules on thesurface of the targeted particles were modified with Tf(adamantane-PEG-Tf). The particles were suspended in a 5% (wt/vol)glucose carrier solution for injection.

Davis et al. (Nature, Vol 464, 15 Apr. 2010) conducts a RNA clinicaltrial that uses a targeted particle-delivery system (clinical trialregistration number NCT00689065). Patients with solid cancers refractoryto standard-of-care therapies are administered doses of targetedparticles on days 1, 3, 8 and 10 of a 21-day cycle by a 30-minintravenous infusion. The particles comprise, consist essentially of, orconsist of a synthetic delivery system containing: (1) a linear,cyclodextrin-based polymer (CDP), (2) a human transferrin protein (TF)targeting ligand displayed on the exterior of the nanoparticle to engageTF receptors (TFR) on the surface of the cancer cells, (3) a hydrophilicpolymer (polyethylene glycol (PEG) used to promote nanoparticlestability in biological fluids), and (4) siRNA designed to reduce theexpression of the RRM2 (sequence used in the clinic was previouslydenoted siR2B+5). The TFR has long been known to be upregulated inmalignant cells, and RRM2 is an established anti-cancer target. Theseparticles (clinical version denoted as CALAA-01) have been shown to bewell tolerated in multi-dosing studies in non human primates. Although asingle patient with chronic myeloid leukaemia has been administeredsiRNA by liposomal delivery, Davis et al.'s clinical trial is theinitial human trial to systemically deliver siRNA with a targeteddelivery system and to treat patients with solid cancer. To ascertainwhether the targeted delivery system can provide effective delivery offunctional siRNA to human tumours, Davis et al. investigated biopsiesfrom three patients from three different dosing cohorts; patients A, Band C, all of whom had metastatic melanoma and received CALAA-01 dosesof 18, 24 and 30 mg m⁻² siRNA, respectively. Similar doses may also becontemplated for the nucleic acid-targeting system of the presentinvention. The delivery of the invention may be achieved with particlescontaining a linear, cyclodextrin-based polymer (CDP), a humantransferrin protein (TF) targeting ligand displayed on the exterior ofthe particle to engage TF receptors (TFR) on the surface of the cancercells and/or a hydrophilic polymer (for example, polyethylene glycol(PEG) used to promote particle stability in biological fluids).

In terms of this invention, it is preferred to have one or morecomponents of RNA-targeting complex, e.g., nucleic acid-targetingeffector (Cas13b) protein or mRNA therefor, or guide RNA or crRNAdelivered using particles or lipid envelopes. Other delivery systems orvectors are may be used in conjunction with the particle aspects of theinvention. Particles encompassed in the present invention may beprovided in different forms, e.g., as solid particles (e.g., metal suchas silver, gold, iron, titanium), non-metal, lipid-based solids,polymers), suspensions of particles, or combinations thereof. Metal,dielectric, and semiconductor particles may be prepared, as well ashybrid structures (e.g., core-shell particles). Particles made ofsemiconducting material may also be labeled quantum dots if they aresmall enough (typically sub 10 nm) that quantization of electronicenergy levels occurs. Such nanoscale particles are used in biomedicalapplications as drug carriers or imaging agents and may be adapted forsimilar purposes in the present invention.

Semi-solid and soft particles have been manufactured, and are within thescope of the present invention. A prototype particle of semi-solidnature is the liposome. Various types of liposome particles arecurrently used clinically as delivery systems for anticancer drugs andvaccines. Particles with one half hydrophilic and the other halfhydrophobic are termed Janus particles and are particularly effectivefor stabilizing emulsions. They can self-assemble at water/oilinterfaces and act as solid surfactants.

U.S. Pat. No. 8,709,843, incorporated herein by reference, provides adrug delivery system for targeted delivery of therapeuticagent-containing particles to tissues, cells, and intracellularcompartments. The invention provides targeted particles comprisingpolymer conjugated to a surfactant, hydrophilic polymer or lipid. U.S.Pat. No. 6,007,845, incorporated herein by reference, provides particleswhich have a core of a multiblock copolymer formed by covalently linkinga multifunctional compound with one or more hydrophobic polymers and oneor more hydrophilic polymers, and contain a biologically activematerial. U.S. Pat. No. 5,855,913, incorporated herein by reference,provides a particulate composition having aerodynamically lightparticles having a tap density of less than 0.4 g/cm3 with a meandiameter of between 5 μm and 30 μm, incorporating a surfactant on thesurface thereof for drug delivery to the pulmonary system. U.S. Pat. No.5,985,309, incorporated herein by reference, provides particlesincorporating a surfactant and/or a hydrophilic or hydrophobic complexof a positively or negatively charged therapeutic or diagnostic agentand a charged molecule of opposite charge for delivery to the pulmonarysystem. U.S. Pat. No. 5,543,158, incorporated herein by reference,provides biodegradable injectable particles having a biodegradable solidcore containing a biologically active material and poly(alkylene glycol)moieties on the surface. WO 2012/135025 (also published asUS20120251560), incorporated herein by reference, describes conjugatedpolyethyleneimine (PEI) polymers and conjugated aza-macrocycles(collectively referred to as “conjugated lipomer” or “lipomers”). Incertain embodiments, it can be envisioned that such methods andmaterials of herein-cited documents, e.g., conjugated lipomers can beused in the context of the nucleic acid-targeting system to achieve invitro, ex vivo and in vivo genomic perturbations to modify geneexpression, including modulation of protein expression.

Exosomes

Exosomes are endogenous nano-vesicles that transport RNAs and proteins,and which can deliver RNA to the brain and other target organs. Toreduce immunogenicity, Alvarez-Erviti et al. (2011, Nat Biotechnol 29:341) used self-derived dendritic cells for exosome production. Targetingto the brain was achieved by engineering the dendritic cells to expressLamp2b, an exosomal membrane protein, fused to the neuron-specific RVGpeptide. Purified exosomes were loaded with exogenous RNA byelectroporation. Intravenously injected RVG-targeted exosomes deliveredGAPDH siRNA specifically to neurons, microglia, oligodendrocytes in thebrain, resulting in a specific gene knockdown. Pre-exposure to RVGexosomes did not attenuate knockdown, and non-specific uptake in othertissues was not observed. The therapeutic potential of exosome-mediatedsiRNA delivery was demonstrated by the strong mRNA (60%) and protein(62%) knockdown of BACE1, a therapeutic target in Alzheimer's disease.

To obtain a pool of immunologically inert exosomes, Alvarez-Erviti etal. harvested bone marrow from inbred C57BL/6 mice with a homogenousmajor histocompatibility complex (MHC) haplotype. As immature dendriticcells produce large quantities of exosomes devoid of T-cell activatorssuch as MHC-II and CD86, Alvarez-Erviti et al. selected for dendriticcells with granulocyte/macrophage-colony stimulating factor (GM-CSF) for7 d. Exosomes were purified from the culture supernatant the followingday using well-established ultracentrifugation protocols. The exosomesproduced were physically homogenous, with a size distribution peaking at80 nm in diameter as determined by particle tracking analysis (NTA) andelectron microscopy. Alvarez-Erviti et al. obtained 6-12 μg of exosomes(measured based on protein concentration) per 10⁶ cells. Next,Alvarez-Erviti et al. investigated the possibility of loading modifiedexosomes with exogenous cargoes using electroporation protocols adaptedfor nanoscale applications. As electroporation for membrane particles atthe nanometer scale is not well-characterized, nonspecific Cy5-labeledRNA was used for the empirical optimization of the electroporationprotocol. The amount of encapsulated RNA was assayed afterultracentrifugation and lysis of exosomes. Electroporation at 400 V and125 μF resulted in the greatest retention of RNA and was used for allsubsequent experiments. Alvarez-Erviti et al. administered 150 μg ofeach BACE1 siRNA encapsulated in 150 μg of RVG exosomes to normalC57BL/6 mice and compared the knockdown efficiency to four controls:untreated mice, mice injected with RVG exosomes only, mice injected withBACE1 siRNA complexed to an in vivo cationic liposome reagent and miceinjected with BACE1 siRNA complexed to RVG-9R, the RVG peptideconjugated to 9 D-arginines that electrostatically binds to the siRNA.Cortical tissue samples were analyzed 3 d after administration and asignificant protein knockdown (45%, P<0.05, versus 62%, P<0.01) in bothsiRNA-RVG-9R-treated and siRNARVG exosome-treated mice was observed,resulting from a significant decrease in BACE1 mRNA levels (66% [+ or −]15%, P<0.001 and 61% [+ or −] 13% respectively, P<0.01). Moreover,Applicants demonstrated a significant decrease (55%, P<0.05) in thetotal [beta]-amyloid 1-42 levels, a main component of the amyloidplaques in Alzheimer's pathology, in the RVG-exosome-treated animals.The decrease observed was greater than the β-amyloid 1-40 decreasedemonstrated in normal mice after intraventricular injection of BACE1inhibitors. Alvarez-Erviti et al. carried out 5′-rapid amplification ofcDNA ends (RACE) on BACE1 cleavage product, which provided evidence ofRNAi-mediated knockdown by the siRNA. Finally, Alvarez-Erviti et al.investigated whether RNA-RVG exosomes induced immune responses in vivoby assessing IL-6, IP-10, TNFα and IFN-α serum concentrations. Followingexosome treatment, nonsignificant changes in all cytokines wereregistered similar to siRNA-transfection reagent treatment in contrastto siRNA-RVG-9R, which potently stimulated IL-6 secretion, confirmingthe immunologically inert profile of the exosome treatment. Given thatexosomes encapsulate only 20% of siRNA, delivery with RVG-exosomeappears to be more efficient than RVG-9R delivery as comparable mRNAknockdown and greater protein knockdown was achieved with fivefold lesssiRNA without the corresponding level of immune stimulation. Thisexperiment demonstrated the therapeutic potential of RVG-exosometechnology, which is potentially suited for long-term silencing of genesrelated to neurodegenerative diseases. The exosome delivery system ofAlvarez-Erviti et al. may be applied to deliver the nucleicacid-targeting system of the present invention to therapeutic targets,especially neurodegenerative diseases. A dosage of about 100 to 1000 mgof nucleic acid-targeting system encapsulated in about 100 to 1000 mg ofRVG exosomes may be contemplated for the present invention.

El-Andaloussi et al. (Nature Protocols 7,2112-2126(2012)) providesexosomes derived from cultured cells harnessed for delivery of RNA invitro and in vivo. This protocol first describes the generation oftargeted exosomes through transfection of an expression vector,comprising an exosomal protein fused with a peptide ligand. Next,El-Andaloussi et al. explain how to purify and characterize exosomesfrom transfected cell supernatant. Next, El-Andaloussi et al. detailcrucial steps for loading RNA into exosomes. Finally, El-Andaloussi etal. outline how to use exosomes to efficiently deliver RNA in vitro andin vivo in mouse brain. Examples of anticipated results in whichexosome-mediated RNA delivery is evaluated by functional assays andimaging are also provided. The entire protocol takes ˜3 weeks. Deliveryor administration according to the invention may be performed usingexosomes produced from self-derived dendritic cells. From the hereinteachings, this can be employed in the practice of the invention.

In another embodiment, the plasma exosomes of Wahlgren et al. (NucleicAcids Research, 2012, Vol. 40, No. 17 e130) are contemplated. Exosomesare nano-sized vesicles (30-90 nm in size) produced by many cell types,including dendritic cells (DC), B cells, T cells, mast cells, epithelialcells and tumor cells. These vesicles are formed by inward budding oflate endosomes and are then released to the extracellular environmentupon fusion with the plasma membrane. Because exosomes naturally carryRNA between cells, this property may be useful in gene therapy, and fromthis disclosure can be employed in the practice of the instantinvention. Exosomes from plasma can be prepared by centrifugation ofbuffy coat at 900 g for 20 min to isolate the plasma followed byharvesting cell supernatants, centrifuging at 300 g for 10 min toeliminate cells and at 16500×g for 30 min followed by filtration througha 0.22 mm filter. Exosomes are pelleted by ultracentrifugation at120000×g for 70 min. Chemical transfection of siRNA into exosomes iscarried out according to the manufacturer's instructions in RNAiHuman/Mouse Starter Kit (Quiagen, Hilden, Germany). siRNA is added to100 ml PBS at a final concentration of 2 mmol/ml. After adding HiPerFecttransfection reagent, the mixture is incubated for 10 min at RT. Inorder to remove the excess of micelles, the exosomes are re-isolatedusing aldehyde/sulfate latex beads. The chemical transfection of nucleicacid-targeting system into exosomes may be conducted similarly to siRNA.The exosomes may be co-cultured with monocytes and lymphocytes isolatedfrom the peripheral blood of healthy donors. Therefore, it may becontemplated that exosomes containing nucleic acid-targeting system maybe introduced to monocytes and lymphocytes of and autologouslyreintroduced into a human. Accordingly, delivery or administrationaccording to the invention may be performed using plasma exosomes.

Liposomes

Delivery or administration according to the invention can be performedwith liposomes. Liposomes are spherical vesicle structures composed of auni- or multilamellar lipid bilayer surrounding internal aqueouscompartments and a relatively impermeable outer lipophilic phospholipidbilayer. Liposomes have gained considerable attention as drug deliverycarriers because they are biocompatible, nontoxic, can deliver bothhydrophilic and lipophilic drug molecules, protect their cargo fromdegradation by plasma enzymes, and transport their load acrossbiological membranes and the blood brain barrier (BBB) (see, e.g., Spuchand Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12pages, 2011. doi:10.1155/2011/469679 for review). Liposomes can be madefrom several different types of lipids; however, phospholipids are mostcommonly used to generate liposomes as drug carriers. Although liposomeformation is spontaneous when a lipid film is mixed with an aqueoussolution, it can also be expedited by applying force in the form ofshaking by using a homogenizer, sonicator, or an extrusion apparatus(see, e.g., Spuch and Navarro, Journal of Drug Delivery, vol. 2011,Article ID 469679, 12 pages, 2011. doi:10.1155/2011/469679 for review).

Several other additives may be added to liposomes in order to modifytheir structure and properties. For instance, either cholesterol orsphingomyelin may be added to the liposomal mixture in order to helpstabilize the liposomal structure and to prevent the leakage of theliposomal inner cargo. Further, liposomes are prepared from hydrogenatedegg phosphatidylcholine or egg phosphatidylcholine, cholesterol, anddicetyl phosphate, and their mean vesicle sizes were adjusted to about50 and 100 nm. (see, e.g., Spuch and Navarro, Journal of Drug Delivery,vol. 2011, Article ID 469679, 12 pages, 2011. doi:10.1155/2011/469679for review). A liposome formulation may be mainly comprised of naturalphospholipids and lipids such as1,2-distearoyl-sn-glycero-3-phosphatidyl choline (DSPC), sphingomyelin,egg phosphatidylcholines and monosialoganglioside. Since thisformulation is made up of phospholipids only, liposomal formulationshave encountered many challenges, one of the ones being the instabilityin plasma. Several attempts to overcome these challenges have been made,specifically in the manipulation of the lipid membrane. One of theseattempts focused on the manipulation of cholesterol. Addition ofcholesterol to conventional formulations reduces rapid release of theencapsulated bioactive compound into the plasma or1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) increases thestability (see, e.g., Spuch and Navarro, Journal of Drug Delivery, vol.2011, Article ID 469679, 12 pages, 2011. doi:10.1155/2011/469679 forreview). In a particularly advantageous embodiment, Trojan Horseliposomes (also known as Molecular Trojan Horses) are desirable andprotocols may be found athttp://cshprotocols/cshlp.org/content/2010/4/pdb.protS407.long. Theseparticles allow delivery of a transgene to the entire brain after anintravascular injection. Without being bound by limitation, it isbelieved that neutral lipid particles with specific antibodiesconjugated to surface allow crossing of the blood brain barrier viaendocytosis. Applicant postulates utilizing Trojan Horse Liposomes todeliver the CRISPR-Cas13b complexes to the brain via an intravascularinjection, which would allow whole brain transgenic animals without theneed for embryonic manipulation. About 1-5 g of DNA or RNA may becontemplated for in vivo administration in liposomes.

In another embodiment, the nucleic acid-targeting system or componentsthereof may be administered in liposomes, such as a stablenucleic-acid-lipid particle (SNALP) (see, e.g., Morrissey et al., NatureBiotechnology, Vol. 23, No. 8, August 2005). Daily intravenousinjections of about 1, 3 or 5 mg/kg/day of a specific nucleicacid-targeting system targeted in a SNALP are contemplated. The dailytreatment may be over about three days and then weekly for about fiveweeks. In another embodiment, a specific nucleic acid-targeting systemencapsulated SNALP) administered by intravenous injection to at doses ofabout 1 or 2.5 mg/kg are also contemplated (see, e.g., Zimmerman et al.,Nature Letters, Vol. 441, 4 May 2006). The SNALP formulation may containthe lipids 3-N-[(methoxypoly(ethylene glycol) 2000)carbamoyl]-1,2-dimyristyloxy-propylamine (PEG-C-DMA),1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA),1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol, in a2:40:10:48 molar percent ratio (see, e.g., Zimmerman et al., NatureLetters, Vol. 441, 4 May 2006). In another embodiment, stablenucleic-acid-lipid particles (SNALPs) have proven to be effectivedelivery molecules to highly vascularized HepG2-derived liver tumors butnot in poorly vascularized HCT-116 derived liver tumors (see, e.g., Li,Gene Therapy (2012) 19, 775-780). The SNALP liposomes may be prepared byformulating D-Lin-DMA and PEG-C-DMA with distearoylphosphatidylcholine(DSPC), Cholesterol and siRNA using a 25:1 lipid/siRNA ratio and a48/40/10/2 molar ratio of Cholesterol/D-Lin-DMA/DSPC/PEG-C-DMA. Theresulted SNALP liposomes are about 80-100 nm in size. In yet anotherembodiment, a SNALP may comprise synthetic cholesterol (Sigma-Aldrich,St Louis, MO, USA), dipalmitoylphosphatidylcholine (Avanti Polar Lipids,Alabaster, AL, USA), 3-N-[(w-methoxy poly(ethyleneglycol)2000)carbamoyl]-1,2-dimyrestyloxypropylamine, and cationic1,2-dilinoleyloxy-3-N,N-dimethylaminopropane (see, e.g., Geisbert etal., Lancet 2010; 375: 1896-905). A dosage of about 2 mg/kg totalnucleic acid-targeting systemper dose administered as, for example, abolus intravenous infusion may be contemplated. In yet anotherembodiment, a SNALP may comprise synthetic cholesterol (Sigma-Aldrich),1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC; Avanti Polar LipidsInc.), PEG-cDMA, and 1,2-dilinoleyloxy-3-(N;N-dimethyl)aminopropane(DLinDMA) (see, e.g., Judge, J. Clin. Invest. 119:661-673 (2009)).Formulations used for in vivo studies may comprise a final lipid/RNAmass ratio of about 9:1.

The safety profile of RNAi nanomedicines has been reviewed by Barros andGollob of Alnylam Pharmaceuticals (see, e.g., Advanced Drug DeliveryReviews 64 (2012) 1730-1737). The stable nucleic acid lipid particle(SNALP) is comprised of four different lipids an ionizable lipid(DLinDMA) that is cationic at low pH, a neutral helper lipid,cholesterol, and a diffusible polyethylene glycol (PEG)-lipid. Theparticle is approximately 80 nm in diameter and is charge-neutral atphysiologic pH. During formulation, the ionizable lipid serves tocondense lipid with the anionic RNA during particle formation. Whenpositively charged under increasingly acidic endosomal conditions, theionizable lipid also mediates the fusion of SNALP with the endosomalmembrane enabling release of RNA into the cytoplasm. The PEG-lipidstabilizes the particle and reduces aggregation during formulation, andsubsequently provides a neutral hydrophilic exterior that improvespharmacokinetic properties. To date, two clinical programs have beeninitiated using SNALP formulations with RNA. Tekmira Pharmaceuticalsrecently completed a phase I single-dose study of SNALP-ApoB in adultvolunteers with elevated LDL cholesterol. ApoB is predominantlyexpressed in the liver and jejunum and is essential for the assembly andsecretion of VLDL and LDL. Seventeen subjects received a single dose ofSNALP-ApoB (dose escalation across 7 dose levels). There was no evidenceof liver toxicity (anticipated as the potential dose-limiting toxicitybased on preclinical studies). One (of two) subjects at the highest doseexperienced flu-like symptoms consistent with immune system stimulation,and the decision was made to conclude the trial. Alnylam Pharmaceuticalshas similarly advanced ALN-TTR01, which employs the SNALP technologydescribed above and targets hepatocyte production of both mutant andwild-type TTR to treat TTR amyloidosis (ATTR). Three ATTR syndromes havebeen described: familial amyloidotic polyneuropathy (FAP) and familialamyloidotic cardiomyopathy (FAC) both caused by autosomal dominantmutations in TTR; and senile systemic amyloidosis (SSA) cause bywildtype TTR. A placebo-controlled, single dose-escalation phase I trialof ALN-TTR01 was recently completed in patients with ATTR. ALN-TTR01 wasadministered as a 15-minute IV infusion to 31 patients (23 with studydrug and 8 with placebo) within a dose range of 0.01 to 1.0 mg/kg (basedon siRNA). Treatment was well tolerated with no significant increases inliver function tests. Infusion-related reactions were noted in 3 of 23patients at ≥0.4 mg/kg; all responded to slowing of the infusion rateand all continued on study. Minimal and transient elevations of serumcytokines IL-6, IP-10 and IL-1ra were noted in two patients at thehighest dose of 1 mg/kg (as anticipated from preclinical and NHPstudies). Lowering of serum TTR, the expected pharmacodynamics effect ofALN-TTR01, was observed at 1 mg/kg.

In yet another embodiment, a SNALP may be made by solubilizing acationic lipid, DSPC, cholesterol and PEG-lipid e.g., in ethanol, e.g.,at a molar ratio of 40:10:40:10, respectively (see, Semple et al.,Nature Biotechnology, Volume 28 Number 2 Feb. 2010, pp. 172-177). Thelipid mixture was added to an aqueous buffer (50 mM citrate, pH 4) withmixing to a final ethanol and lipid concentration of 30% (vol/vol) and6.1 mg/ml, respectively, and allowed to equilibrate at 22° C. for 2 minbefore extrusion. The hydrated lipids were extruded through two stacked80 nm pore-sized filters (Nuclepore) at 22° C. using a Lipex Extruder(Northern Lipids) until a vesicle diameter of 70-90 nm, as determined bydynamic light scattering analysis, was obtained. This generally required1-3 passes. The siRNA (solubilized in a 50 mM citrate, pH 4 aqueoussolution containing 30% ethanol) was added to the pre-equilibrated (35°C.) vesicles at a rate of ˜5 ml/min with mixing. After a final targetsiRNA/lipid ratio of 0.06 (wt/wt) was reached, the mixture was incubatedfor a further 30 min at 35° C. to allow vesicle reorganization andencapsulation of the siRNA. The ethanol was then removed and theexternal buffer replaced with PBS (155 mM NaCl, 3 mM Na₂HPO₄, 1 mMKH₂PO₄, pH 7.5) by either dialysis or tangential flow diafiltration.siRNA were encapsulated in SNALP using a controlled step-wise dilutionmethod process. The lipid constituents of KC2-SNALP were DLin-KC2-DMA(cationic lipid), dipalmitoylphosphatidylcholine (DPPC; Avanti PolarLipids), synthetic cholesterol (Sigma) and PEG-C-DMA used at a molarratio of 57.1:7.1:34.3:1.4. Upon formation of the loaded particles,SNALP were dialyzed against PBS and filter sterilized through a 0.2 μmfilter before use. Mean particle sizes were 75-85 nm and 90-95% of thesiRNA was encapsulated within the lipid particles. The final siRNA/lipidratio in formulations used for in vivo testing was ˜0.15 (wt/wt).LNP-siRNA systems containing Factor VII siRNA were diluted to theappropriate concentrations in sterile PBS immediately before use and theformulations were administered intravenously through the lateral tailvein in a total volume of 10 ml/kg. This method and these deliverysystems may be extrapolated to the nucleic acid-targeting system of thepresent invention.

Other Lipids

Other cationic lipids, such as amino lipid2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA) maybe utilized to encapsulate nucleic acid-targeting system or componentsthereof or nucleic acid molecule(s) coding therefor e.g., similar toSiRNA (see, e.g., Jayaraman, Angew. Chem. Int. Ed. 2012, 51, 8529-8533),and hence may be employed in the practice of the invention. A preformedvesicle with the following lipid composition may be contemplated: aminolipid, distearoylphosphatidylcholine (DSPC), cholesterol and (R)-2,3-bis(octadecyloxy) propyl-1-(methoxy poly(ethyleneglycol)2000)propylcarbamate (PEG-lipid) in the molar ratio 40/10/40/10,respectively, and a FVII siRNA/total lipid ratio of approximately 0.05(w/w). To ensure a narrow particle size distribution in the range of70-90 nm and a low polydispersity index of 0.11+0.04 (n=56), theparticles may be extruded up to three times through 80 nm membranesprior to adding the guide RNA. Particles containing the highly potentamino lipid 16 may be used, in which the molar ratio of the four lipidcomponents 16, DSPC, cholesterol and PEG-lipid (50/10/38.5/1.5) whichmay be further optimized to enhance in vivo activity.

Michael S D Kormann et al. (“Expression of therapeutic proteins afterdelivery of chemically modified mRNA in mice: Nature Biotechnology,Volume:29, Pages: 154-157 (2011)) describes the use of lipid envelopesto deliver RNA. Use of lipid envelopes is also preferred in the presentinvention.

In another embodiment, lipids may be formulated with the RNA-targetingsystem (CRISPR-Cas13b complex, i.e., the Cas13b complexed with crRNA) ofthe present invention or component(s) thereof or nucleic acidmolecule(s) coding therefor to form lipid nanoparticles (LNPs). Lipidsinclude, but are not limited to, DLin-KC2-DMA4, C12-200 and colipidsdistearoylphosphatidyl choline, cholesterol, and PEG-DMG may beformulated with RNA-targeting system instead of siRNA (see, e.g.,Novobrantseva, Molecular Therapy-Nucleic Acids (2012) 1, e4;doi:10.1038/mtna.2011.3) using a spontaneous vesicle formationprocedure. The component molar ratio may be about 50/10/38.5/1.5(DLin-KC2-DMA or C12-200/distearoylphosphatidylcholine/cholesterol/PEG-DMG). The final lipid: siRNA weight ratio may be˜12:1 and 9:1 in the case of DLin-KC2-DMA and C12-200 lipid particles(LNPs), respectively. The formulations may have mean particle diametersof ˜80 nm with >90% entrapment efficiency. A 3 mg/kg dose may becontemplated. Tekmira has a portfolio of approximately 95 patentfamilies, in the U.S. and abroad, that are directed to various aspectsof LNPs and LNP formulations (see, e.g., U.S. Pat. Nos. 7,982,027;7,799,565; 8,058,069; 8,283,333; 7,901,708; 7,745,651; 7,803,397;8,101,741; 8,188,263; 7,915,399; 8,236,943 and 7,838,658 and EuropeanPat. Nos 1766035; 1519714; 1781593 and 1664316), all of which may beused and/or adapted to the present invention.

The RNA-targeting system or components thereof or nucleic acidmolecule(s) coding therefor may be delivered encapsulated in PLGAMicrospheres such as that further described in US published applications20130252281 and 20130245107 and 20130244279 (assigned to ModernaTherapeutics) which relate to aspects of formulation of compositionscomprising modified nucleic acid molecules which may encode a protein, aprotein precursor, or a partially or fully processed form of the proteinor a protein precursor. The formulation may have a molar ratio50:10:38.5:1.5-3.0 (cationic lipid:fusogenic lipid:cholesterol:PEGlipid). The PEG lipid may be selected from, but is not limited toPEG-c-DOMG, PEG-DMG. The fusogenic lipid may be DSPC. See also, Schrumet al., Delivery and Formulation of Engineered Nucleic Acids, USpublished application 20120251618.

Nanomerics' technology addresses bioavailability challenges for a broadrange of therapeutics, including low molecular weight hydrophobic drugs,peptides, and nucleic acid based therapeutics (plasmid, siRNA, miRNA).Specific administration routes for which the technology has demonstratedclear advantages include the oral route, transport across theblood-brain-barrier, delivery to solid tumours, as well as to the eye.See, e.g., Mazza et al., 2013, ACS Nano. 2013 Feb. 26; 7(2):1016-26;Uchegbu and Siew, 2013, J Pharm Sci. 102(2):305-10 and Lalatsa et al.,2012, J Control Release. 2012 Jul. 20; 161(2):523-36.

US Patent Publication No. 20050019923 describes cationic dendrimers fordelivering bioactive molecules, such as polynucleotide molecules,peptides and polypeptides and/or pharmaceutical agents, to a mammalianbody. The dendrimers are suitable for targeting the delivery of thebioactive molecules to, for example, the liver, spleen, lung, kidney orheart (or even the brain). Dendrimers are synthetic 3-dimensionalmacromolecules that are prepared in a step wise fashion from simplebranched monomer units, the nature and functionality of which can beeasily controlled and varied. Dendrimers are synthesized from therepeated addition of building blocks to a multifunctional core(divergent approach to synthesis), or towards a multifunctional core(convergent approach to synthesis) and each addition of a 3-dimensionalshell of building blocks leads to the formation of a higher generationof the dendrimers. Polypropylenimine dendrimers start from adiaminobutane core to which is added twice the number of amino groups bya double Michael addition of acrylonitrile to the primary aminesfollowed by the hydrogenation of the nitriles. This results in adoubling of the amino groups. Polypropylenimine dendrimers contain 100%protonable nitrogens and up to 64 terminal amino groups (generation 5,DAB 64). Protonable groups are usually amine groups which are able toaccept protons at neutral pH. The use of dendrimers as gene deliveryagents has largely focused on the use of the polyamidoamine. andphosphorous containing compounds with a mixture of amine/amide orN—P(O₂)S as the conjugating units respectively with no work beingreported on the use of the lower generation polypropylenimine dendrimersfor gene delivery. Polypropylenimine dendrimers have also been studiedas pH sensitive controlled release systems for drug delivery and fortheir encapsulation of guest molecules when chemically modified byperipheral amino acid groups. The cytotoxicity and interaction ofpolypropylenimine dendrimers with DNA as well as the transfectionefficacy of DAB 64 has also been studied. US Patent Publication No.20050019923 is based upon the observation that, contrary to earlierreports, cationic dendrimers, such as polypropylenimine dendrimers,display suitable properties, such as specific targeting and lowtoxicity, for use in the targeted delivery of bioactive molecules, suchas genetic material. In addition, derivatives of the cationic dendrimeralso display suitable properties for the targeted delivery of bioactivemolecules. See also, Bioactive Polymers, US published application20080267903, which discloses “Various polymers, including cationicpolyamine polymers and dendrimeric polymers, are shown to possessanti-proliferative activity, and may therefore be useful for treatmentof disorders characterised by undesirable cellular proliferation such asneoplasms and tumours, inflammatory disorders (including autoimmunedisorders), psoriasis and atherosclerosis. The polymers may be usedalone as active agents, or as delivery vehicles for other therapeuticagents, such as drug molecules or nucleic acids for gene therapy. Insuch cases, the polymers' own intrinsic anti-tumour activity maycomplement the activity of the agent to be delivered.” The disclosuresof these patent publications may be employed in conjunction with hereinteachings for delivery of nucleic acid-targetingsystem(s) orcomponent(s) thereof or nucleic acid molecule(s) coding therefor.

Supercharged Proteins

Supercharged proteins are a class of engineered or naturally occurringproteins with unusually high positive or negative net theoretical chargeand may be employed in delivery of nucleic acid-targetingsystem(s) orcomponent(s) thereof or nucleic acid molecule(s) coding therefor. Bothsupernegatively and superpositively charged proteins exhibit aremarkable ability to withstand thermally or chemically inducedaggregation. Superpositively charged proteins are also able to penetratemammalian cells. Associating cargo with these proteins, such as plasmidDNA, RNA, or other proteins, can enable the functional delivery of thesemacromolecules into mammalian cells both in vitro and in vivo. DavidLiu's lab reported the creation and characterization of superchargedproteins in 2007 (Lawrence et al., 2007, Journal of the AmericanChemical Society 129, 10110-10112).

The nonviral delivery of RNA and plasmid DNA into mammalian cells arevaluable both for research and therapeutic applications (Akinc et al.,2010, Nat. Biotech. 26, 561-569). Purified +36 GFP protein (or othersuperpositively charged protein) is mixed with RNAs in the appropriateserum-free media and allowed to complex prior addition to cells.Inclusion of serum at this stage inhibits formation of the superchargedprotein-RNA complexes and reduces the effectiveness of the treatment.The following protocol has been found to be effective for a variety ofcell lines (McNaughton et al., 2009, Proc. Natl. Acad. Sci. USA 106,6111-6116). However, pilot experiments varying the dose of protein andRNA should be performed to optimize the procedure for specific celllines. (1) One day before treatment, plate 1×10⁵ cells per well in a48-well plate. (2) On the day of treatment, dilute purified +36 GFPprotein in serum free media to a final concentration 200 nM. Add RNA toa final concentration of 50 nM. Vortex to mix and incubate at roomtemperature for 10 min. (3) During incubation, aspirate media from cellsand wash once with PBS. (4) Following incubation of +36 GFP and RNA, addthe protein-RNA complexes to cells. (5) Incubate cells with complexes at37° C. for 4 h. (6) Following incubation, aspirate the media and washthree times with 20 U/mL heparin PBS. Incubate cells withserum-containing media for a further 48 h or longer depending upon theassay for activity. (7) Analyze cells by immunoblot, qPCR, phenotypicassay, or other appropriate method.

+36 GFP was found to be an effective plasmid delivery reagent in a rangeof cells. See also, e.g., McNaughton et al., Proc. Natl. Acad. Sci. USA106, 6111-6116 (2009); Cronican et al., ACS Chemical Biology 5, 747-752(2010); Cronican et al., Chemistry & Biology 18, 833-838 (2011);Thompson et al., Methods in Enzymology 503, 293-319 (2012); Thompson, D.B., et al., Chemistry & Biology 19 (7), 831-843 (2012). The methods ofthe super charged proteins may be used and/or adapted for delivery ofthe RNA-targeting system(s) or component(s) thereof or nucleic acidmolecule(s) coding therefor of the invention.

Cell Penetrating Peptides (CPPs)

In yet another embodiment, cell penetrating peptides (CPPs) arecontemplated for the delivery of the CRISPR Cas system. CPPs are shortpeptides that facilitate cellular uptake of various molecular cargo(from nanosize particles to small chemical molecules and large fragmentsof DNA). The term “cargo” as used herein includes but is not limited tothe group consisting of therapeutic agents, diagnostic probes, peptides,nucleic acids, antisense oligonucleotides, plasmids, proteins, particlesincluding nanoparticles, liposomes, chromophores, small molecules andradioactive materials. In aspects of the invention, the cargo may alsocomprise any component of the CRISPR Cas system or the entire functionalCRISPR Cas system. Aspects of the present invention further providemethods for delivering a desired cargo into a subject comprising: (a)preparing a complex comprising the cell penetrating peptide of thepresent invention and a desired cargo, and (b) orally, intraarticularly,intraperitoneally, intrathecally, intraarterially, intranasally,intraparenchymally, subcutaneously, intramuscularly, intravenously,dermally, intrarectally, or topically administering the complex to asubject. The cargo is associated with the peptides either throughchemical linkage via covalent bonds or through non-covalentinteractions. The function of the CPPs are to deliver the cargo intocells, a process that commonly occurs through endocytosis with the cargodelivered to the endosomes of living mammalian cells. Cell-penetratingpeptides are of different sizes, amino acid sequences, and charges butall CPPs have one distinct characteristic, which is the ability totranslocate the plasma membrane and facilitate the delivery of variousmolecular cargoes to the cytoplasm or an organelle. CPP translocationmay be classified into three main entry mechanisms: direct penetrationin the membrane, endocytosis-mediated entry, and translocation throughthe formation of a transitory structure. CPPs have found numerousapplications in medicine as drug delivery agents in the treatment ofdifferent diseases including cancer and virus inhibitors, as well ascontrast agents for cell labeling. Examples of the latter include actingas a carrier for GFP, MRI contrast agents, or quantum dots. CPPs holdgreat potential as in vitro and in vivo delivery vectors for use inresearch and medicine. CPPs typically have an amino acid compositionthat either contains a high relative abundance of positively chargedamino acids such as lysine or arginine or has sequences that contain analternating pattern of polar/charged amino acids and non-polar,hydrophobic amino acids. These two types of structures are referred toas polycationic or amphipathic, respectively. A third class of CPPs arethe hydrophobic peptides, containing only apolar residues, with low netcharge or have hydrophobic amino acid groups that are crucial forcellular uptake. One of the initial CPPs discovered was thetrans-activating transcriptional activator (Tat) from HumanImmunodeficiency Virus 1 (HIV-1) which was found to be efficiently takenup from the surrounding media by numerous cell types in culture. Sincethen, the number of known CPPs has expanded considerably and smallmolecule synthetic analogues with more effective protein transductionproperties have been generated. CPPs include but are not limited toPenetratin, Tat (48-60), Transportan, and (R-AhX-R4)(Ahx=aminohexanoyl).

U.S. Pat. No. 8,372,951, provides a CPP derived from eosinophil cationicprotein (ECP) which exhibits highly cell-penetrating efficiency and lowtoxicity. Aspects of delivering the CPP with its cargo into a vertebratesubject are also provided. Further aspects of CPPs and their deliveryare described in U.S. Pat. No. 8,575,305; 8; 614,194 and 8,044,019. CPPscan be used to deliver the CRISPR-Cas system or components thereof. ThatCPPs can be employed to deliver the CRISPR-Cas system or componentsthereof is also provided in the manuscript “Gene disruption bycell-penetrating peptide-mediated delivery of Cas9 protein and guideRNA”, by Suresh Ramakrishna, Abu-Bonsrah Kwaku Dad, Jagadish Beloor, etal. Genome Res. 2014 Apr. 2. [Epub ahead of print], incorporated byreference in its entirety, wherein it is demonstrated that treatmentwith CPP-conjugated recombinant Cas9 protein and CPP-complexed guideRNAs lead to endogenous gene disruptions in human cell lines. In thepaper the Cas9 protein was conjugated to CPP via a thioether bond,whereas the guide RNA was complexed with CPP, forming condensed,positively charged particles. It was shown that simultaneous andsequential treatment of human cells, including embryonic stem cells,dermal fibroblasts, HEK293T cells, HeLa cells, and embryonic carcinomacells, with the modified Cas9 and guide RNA led to efficient genedisruptions with reduced off-target mutations relative to plasmidtransfections. CPP delivery can be used in the practice of theinvention.

Implantable Devices

In another embodiment, implantable devices are also contemplated fordelivery of the RNA-targeting system or component(s) thereof or nucleicacid molecule(s) coding therefor. For example, US Patent Publication20110195123 discloses an implantable medical device which elutes a druglocally and in prolonged period is provided, including several types ofsuch a device, the treatment modes of implementation and methods ofimplantation. The device comprising of polymeric substrate, such as amatrix for example, that is used as the device body, and drugs, and insome cases additional scaffolding materials, such as metals oradditional polymers, and materials to enhance visibility and imaging. Animplantable delivery device can be advantageous in providing releaselocally and over a prolonged period, where drug is released directly tothe extracellular matrix (ECM) of the diseased area such as tumor,inflammation, degeneration or for symptomatic objectives, or to injuredsmooth muscle cells, or for prevention. One kind of drug is RNA, asdisclosed above, and this system may be used/and or adapted to thenucleic acid-targeting system of the present invention. The modes ofimplantation in some embodiments are existing implantation proceduresthat are developed and used today for other treatments, includingbrachytherapy and needle biopsy. In such cases the dimensions of the newimplant described in this invention are similar to the original implant.Typically a few devices are implanted during the same treatmentprocedure. US Patent Publication 20110195123, provides a drug deliveryimplantable or insertable system, including systems applicable to acavity such as the abdominal cavity and/or any other type ofadministration in which the drug delivery system is not anchored orattached, comprising a biostable and/or degradable and/or bioabsorbablepolymeric substrate, which may for example optionally be a matrix. Itshould be noted that the term “insertion” also includes implantation.The drug delivery system is preferably implemented as a “Loder” asdescribed in US Patent Publication 20110195123. The polymer or pluralityof polymers are biocompatible, incorporating an agent and/or pluralityof agents, enabling the release of agent at a controlled rate, whereinthe total volume of the polymeric substrate, such as a matrix forexample, in some embodiments is optionally and preferably no greaterthan a maximum volume that permits a therapeutic level of the agent tobe reached. As a non-limiting example, such a volume is preferablywithin the range of 0.1 m³ to 1000 mm³, as required by the volume forthe agent load. The Loder may optionally be larger, for example whenincorporated with a device whose size is determined by functionality,for example and without limitation, a knee joint, an intra-uterine orcervical ring and the like. The drug delivery system (for delivering thecomposition) is designed in some embodiments to preferably employdegradable polymers, wherein the main release mechanism is bulk erosion;or in some embodiments, non degradable, or slowly degraded polymers areused, wherein the main release mechanism is diffusion rather than bulkerosion, so that the outer part functions as membrane, and its internalpart functions as a drug reservoir, which practically is not affected bythe surroundings for an extended period (for example from about a weekto about a few months). Combinations of different polymers withdifferent release mechanisms may also optionally be used. Theconcentration gradient at the surface is preferably maintainedeffectively constant during a significant period of the total drugreleasing period, and therefore the diffusion rate is effectivelyconstant (termed “zero mode” diffusion). By the term “constant” it ismeant a diffusion rate that is preferably maintained above the lowerthreshold of therapeutic effectiveness, but which may still optionallyfeature an initial burst and/or may fluctuate, for example increasingand decreasing to a certain degree. The diffusion rate is preferably somaintained for a prolonged period, and it can be considered constant toa certain level to optimize the therapeutically effective period, forexample the effective silencing period. The drug delivery systemoptionally and preferably is designed to shield the nucleotide basedtherapeutic agent from degradation, whether chemical in nature or due toattack from enzymes and other factors in the body of the subject. Thedrug delivery system of US Patent Publication 20110195123 is optionallyassociated with sensing and/or activation appliances that are operatedat and/or after implantation of the device, by non and/or minimallyinvasive methods of activation and/or acceleration/deceleration, forexample optionally including but not limited to thermal heating andcooling, laser beams, and ultrasonic, including focused ultrasoundand/or RF (radiofrequency) methods or devices. According to someembodiments of US Patent Publication 20110195123, the site for localdelivery may optionally include target sites characterized by highabnormal proliferation of cells, and suppressed apoptosis, includingtumors, active and or chronic inflammation and infection includingautoimmune diseases states, degenerating tissue including muscle andnervous tissue, chronic pain, degenerative sites, and location of bonefractures and other wound locations for enhancement of regeneration oftissue, and injured cardiac, smooth and striated muscle. The site forimplantation of the composition, or target site, preferably features aradius, area and/or volume that is sufficiently small for targeted localdelivery. For example, the target site optionally has a diameter in arange of from about 0.1 mm to about 5 cm. The location of the targetsite is preferably selected for maximum therapeutic efficacy. Forexample, the composition of the drug delivery system (optionally with adevice for implantation as described above) is optionally and preferablyimplanted within or in the proximity of a tumor environment, or theblood supply associated thereof. For example the composition (optionallywith the device) is optionally implanted within or in the proximity topancreas, prostate, breast, liver, via the nipple, within the vascularsystem and so forth. The target location is optionally selected from thegroup comprising, consisting essentially of, or consisting of (asnon-limiting examples only, as optionally any site within the body maybe suitable for implanting a Loder): 1. brain at degenerative sites likein Parkinson or Alzheimer disease at the basal ganglia, white and graymatter; 2. spine as in the case of amyotrophic lateral sclerosis (ALS);3. uterine cervix to prevent HPV infection; 4. active and chronicinflammatory joints; 5. dermis as in the case of psoriasis; 6.sympathetic and sensoric nervous sites for analgesic effect; 7. Intraosseous implantation; 8. acute and chronic infection sites; 9. Intravaginal; 10. Inner ear—auditory system, labyrinth of the inner ear,vestibular system; 11. Intra tracheal; 12. Intra-cardiac; coronary,epicardiac; 13. urinary bladder; 14. biliary system; 15. parenchymaltissue including and not limited to the kidney, liver, spleen; 16. lymphnodes; 17. salivary glands; 18. dental gums; 19. Intra-articular (intojoints); 20. Intra-ocular; 21. Brain tissue; 22. Brain ventricles; 23.Cavities, including abdominal cavity (for example but withoutlimitation, for ovary cancer); 24. Intra esophageal and 25. Intrarectal.

Optionally insertion of the system (for example a device containing thecomposition) is associated with injection of material to the ECM at thetarget site and the vicinity of that site to affect local pH and/ortemperature and/or other biological factors affecting the diffusion ofthe drug and/or drug kinetics in the ECM, of the target site and thevicinity of such a site. Optionally, according to some embodiments, therelease of said agent could be associated with sensing and/or activationappliances that are operated prior and/or at and/or after insertion, bynon and/or minimally invasive and/or else methods of activation and/oracceleration/deceleration, including laser beam, radiation, thermalheating and cooling, and ultrasonic, including focused ultrasound and/orRF (radiofrequency) methods or devices, and chemical activators.

According to embodiments of US Patent Publication 20110195123 that canbe used in the practice of the invention, the drug preferably comprisesa RNA, for example for localized cancer cases in breast, pancreas,brain, kidney, bladder, lung, and prostate as described below. Althoughexemplified with RNAi, many drugs are applicable to be encapsulated inLoder, and can be used in association with this invention, as long assuch drugs can be encapsulated with the Loder substrate, such as amatrix for example, and this system may be used and/or adapted todeliver the nucleic acid-targeting system of the present invention. Asanother example of a specific application, neuro and musculardegenerative diseases develop due to abnormal gene expression. Localdelivery of RNAs may have therapeutic properties for interfering withsuch abnormal gene expression. Local delivery of anti apoptotic, antiinflammatory and anti degenerative drugs including small drugs andmacromolecules may also optionally be therapeutic. In such cases theLoder is applied for prolonged release at constant rate and/or through adedicated device that is implanted separately.

All of this may be used and/or adapted to the RNA-targeting system ofthe present invention. Implantable device technology herein discussedcan be employed with herein teachings and hence by this disclosure andthe knowledge in the art, CRISPR-Cas13b system or complex or componentsthereof or nucleic acid molecules thereof or encoding or providingcomponents may be delivered via an implantable device.

Patient-Specific Screening Methods

A nucleic acid-targeting system that targets RNA can be used to screenpatients or patient samples for the presence of particular RNA.

CRISPR Effector Protein mRNA and Guide RNA

CRISPR effector (Cas13b) protein or mRNA therefor (or more generally anucleuic acid molecule therefor) and guide RNA or crRNA might also bedelivered separately e.g., the former 1-12 hours (preferably around 2-6hours) prior to the administration of guide RNA or crRNA, or together. Asecond booster dose of guide RNA or crRNA can be administered 1-12 hours(preferably around 2-6 hours) after the initial administration.

The Cas13b effector protein is sometimes referred to herein as a CRISPREnzyme. It will be appreciated that the effector protein is based on orderived from an enzyme, so the term ‘effector protein’ certainlyincludes ‘enzyme’ in some embodiments. However, it will also beappreciated that the effector protein may, as required in someembodiments, have DNA or RNA binding, but not necessarily cutting ornicking, activity, including a dead-Cas effector protein function.

Cellular targets include Hemopoietic Stem/Progenitor Cells (CD34+);Human T cells; and Eye (retinal cells)—for example photoreceptorprecursor cells.

Inventive methods can further comprise delivery of templates. Deliveryof templates may be via the cotemporaneous or separate from delivery ofany or all the CRISPR effector protein (Cas13b) or guide or crRNA andvia the same delivery mechanism or different. Inducible

Systems

In some embodiments, a CRISPR effector (Cas 13n) protein may form acomponent of an inducible system. The inducible nature of the systemwould allow for spatiotemporal control of gene editing or geneexpression using a form of energy. The form of energy may include but isnot limited to electromagnetic radiation, sound energy, chemical energyand thermal energy. Examples of inducible system include tetracyclineinducible promoters (Tet-On or Tet-Off), small molecule two-hybridtranscription activations systems (FKBP, ABA, etc), or light induciblesystems (Phytochrome, LOV domains, or cryptochrome). In one embodiment,the CRISPR effector protein may be a part of a Light InducibleTranscriptional Effector (LITE) to direct changes in transcriptionalactivity in a sequence-specific manner. The components of a light mayinclude a CRISPR effector protein, a light-responsive cytochromeheterodimer (e.g. from Arabidopsis thaliana), and a transcriptionalactivation/repression domain. Further examples of inducible DNA bindingproteins and methods for their use are provided in U.S. 61/736,465 andU.S. 61/721,283, and WO 2014/018423 A2 which is hereby incorporated byreference in its entirety.

Self-Inactivating Systems

Once all copies of RNA in a cell have been edited, continued a Cas13beffector protein expression or activity in that cell is no longernecessary. A Self-Inactivating system that relies on the use of RNA asto the Cas13b or crRNA as the guide target sequence can shut down thesystem by preventing expression of Cas13b or complex formation.

Kits

In one aspect, the invention provides kits containing any one or more ofthe elements disclosed in the above methods and compositions. In someembodiments, the kit comprises a vector system as taught herein or oneor more of the components of the CRISPR/Cas13b system or complex astaught herein, such as crRNAs and/or Cas13b effector protein or Cas13beffector protein encoding mRNA, and instructions for using the kit.Elements may be provide individually or in combinations, and may beprovided in any suitable container, such as a vial, a bottle, or a tube.In some embodiments, the kit includes instructions in one or morelanguages, for example in more than one language. The instructions maybe specific to the applications and methods described herein. In someembodiments, a kit comprises one or more reagents for use in a processutilizing one or more of the elements described herein. Reagents may beprovided in any suitable container. For example, a kit may provide oneor more reaction or storage buffers. Reagents may be provided in a formthat is usable in a particular assay, or in a form that requiresaddition of one or more other components before use (e.g., inconcentrate or lyophilized form). A buffer can be any buffer, includingbut not limited to a sodium carbonate buffer, a sodium bicarbonatebuffer, a borate buffer, a Tris buffer, a MOPS buffer, a HEPES buffer,and combinations thereof. In some embodiments, the buffer is alkaline.In some embodiments, the buffer has a pH from about 7 to about 10. Insome embodiments, the kit comprises one or more oligonucleotidescorresponding to a guide sequence for insertion into a vector so as tooperably link the guide or crRNA sequence and a regulatory element. Insome embodiments, the kit comprises a homologous recombination templatepolynucleotide. In some embodiments, the kit comprises one or more ofthe vectors and/or one or more of the polynucleotides described herein.The kit may advantageously allows to provide all elements of the systemsof the invention.

The invention has a broad spectrum of applications in, e.g., genetherapy, drug screening, disease diagnosis, and prognosis.

The terms “polynucleotide”, “nucleotide”, “nucleotide sequence”,“nucleic acid” and “oligonucleotide” are used interchangeably. Theyrefer to a polymeric form of nucleotides of any length, eitherdeoxyribonucleotides or ribonucleotides, or analogs thereof.Polynucleotides may have any three dimensional structure, and mayperform any function, known or unknown. The following are non-limitingexamples of polynucleotides: coding or non-coding regions of a gene orgene fragment, loci (locus) defined from linkage analysis, exons,introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, shortinterfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA),ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides,plasmids, vectors, isolated DNA of any sequence, isolated RNA of anysequence, nucleic acid probes, and primers. The term also encompassesnucleic-acid-like structures with synthetic backbones, see, e.g.,Eckstein, 1991; Baserga et al., 1992; Milligan, 1993; WO 97/03211; WO96/39154; Mata, 1997; Strauss-Soukup, 1997; and Samstag, 1996. Apolynucleotide may comprise one or more modified nucleotides, such asmethylated nucleotides and nucleotide analogs. If present, modificationsto the nucleotide structure may be imparted before or after assembly ofthe polymer. The sequence of nucleotides may be interrupted bynon-nucleotide components. A polynucleotide may be further modifiedafter polymerization, such as by conjugation with a labeling component.As used herein the term “wild type” is a term of the art understood byskilled persons and means the typical form of an organism, strain, geneor characteristic as it occurs in nature as distinguished from mutant orvariant forms. A “wild type” can be a base line. As used herein the term“variant” should be taken to mean the exhibition of qualities that havea pattern that deviates from what occurs in nature. The terms“non-naturally occurring” or “engineered” are used interchangeably andindicate the involvement of the hand of man. The terms, when referringto nucleic acid molecules or polypeptides mean that the nucleic acidmolecule or the polypeptide is at least substantially free from at leastone other component with which they are naturally associated in natureand as found in nature. “Complementarity” refers to the ability of anucleic acid to form hydrogen bond(s) with another nucleic acid sequenceby either traditional Watson-Crick base pairing or other non-traditionaltypes. A percent complementarity indicates the percentage of residues ina nucleic acid molecule which can form hydrogen bonds (e.g.,Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5,6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100%complementary). “Perfectly complementary” means that all the contiguousresidues of a nucleic acid sequence will hydrogen bond with the samenumber of contiguous residues in a second nucleic acid sequence.“Substantially complementary” as used herein refers to a degree ofcomplementarity that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,97%, 98%, 99%, or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or morenucleotides, or refers to two nucleic acids that hybridize understringent conditions. As used herein, “stringent conditions” forhybridization refer to conditions under which a nucleic acid havingcomplementarity to a target sequence predominantly hybridizes with thetarget sequence, and substantially does not hybridize to non-targetsequences. Stringent conditions are generally sequence-dependent, andvary depending on a number of factors. In general, the longer thesequence, the higher the temperature at which the sequence specificallyhybridizes to its target sequence. Non-limiting examples of stringentconditions are described in detail in Tijssen (1993), LaboratoryTechniques In Biochemistry And Molecular Biology-Hybridization WithNucleic Acid Probes Part I, Second Chapter “Overview of principles ofhybridization and the strategy of nucleic acid probe assay”, Elsevier,N.Y. Where reference is made to a polynucleotide sequence, thencomplementary or partially complementary sequences are also envisaged.These are preferably capable of hybridizing to the reference sequenceunder highly stringent conditions. Generally, in order to maximize thehybridization rate, relatively low-stringency hybridization conditionsare selected: about 20 to 25° C. lower than the thermal melting point(T_(m)). The T_(m) is the temperature at which 50% of specific targetsequence hybridizes to a perfectly complementary probe in solution at adefined ionic strength and pH. Generally, in order to require at leastabout 85% nucleotide complementarity of hybridized sequences, highlystringent washing conditions are selected to be about 5 to 15° C. lowerthan the T. In order to require at least about 70% nucleotidecomplementarity of hybridized sequences, moderately-stringent washingconditions are selected to be about 15 to 30° C. lower than the T.Highly permissive (very low stringency) washing conditions may be as lowas 50° C. below the T_(m), allowing a high level of mis-matching betweenhybridized sequences. Those skilled in the art will recognize that otherphysical and chemical parameters in the hybridization and wash stagescan also be altered to affect the outcome of a detectable hybridizationsignal from a specific level of homology between target and probesequences. Preferred highly stringent conditions comprise incubation in50% formamide, 5×SSC, and 1% SDS at 42° C., or incubation in 5×SSC and1% SDS at 65° C., with wash in 0.2×SSC and 0.1% SDS at 65° C.“Hybridization” refers to a reaction in which one or morepolynucleotides react to form a complex that is stabilized via hydrogenbonding between the bases of the nucleotide residues. The hydrogenbonding may occur by Watson Crick base pairing, Hoogsteen binding, or inany other sequence specific manner. The complex may comprise two strandsforming a duplex structure, three or more strands forming a multistranded complex, a single self-hybridizing strand, or any combinationof these. A hybridization reaction may constitute a step in a moreextensive process, such as the initiation of PCR, or the cleavage of apolynucleotide by an enzyme. A sequence capable of hybridizing with agiven sequence is referred to as the “complement” of the given sequence.As used herein, the term “genomic locus” or “locus” (plural loci) is thespecific location of a gene or DNA sequence on a chromosome. A “gene”refers to stretches of DNA or RNA that encode a polypeptide or an RNAchain that has functional role to play in an organism and hence is themolecular unit of heredity in living organisms. For the purpose of thisinvention it may be considered that genes include regions which regulatethe production of the gene product, whether or not such regulatorysequences are adjacent to coding and/or transcribed sequences.Accordingly, a gene includes, but is not necessarily limited to,promoter sequences, terminators, translational regulatory sequences suchas ribosome binding sites and internal ribosome entry sites, enhancers,silencers, insulators, boundary elements, replication origins, matrixattachment sites and locus control regions. As used herein, “expressionof a genomic locus” or “gene expression” is the process by whichinformation from a gene is used in the synthesis of a functional geneproduct. The products of gene expression are often proteins, but innon-protein coding genes such as rRNA genes or tRNA genes, the productis functional RNA. The process of gene expression is used by all knownlife—eukaryotes (including multicellular organisms), prokaryotes(bacteria and archaea) and viruses to generate functional products tosurvive. As used herein “expression” of a gene or nucleic acidencompasses not only cellular gene expression, but also thetranscription and translation of nucleic acid(s) in cloning systems andin any other context. As used herein, “expression” also refers to theprocess by which a polynucleotide is transcribed from a DNA template(such as into and mRNA or other RNA transcript) and/or the process bywhich a transcribed mRNA is subsequently translated into peptides,polypeptides, or proteins. Transcripts and encoded polypeptides may becollectively referred to as “gene product.” If the polynucleotide isderived from genomic DNA, expression may include splicing of the mRNA ina eukaryotic cell. The terms “polypeptide”, “peptide” and “protein” areused interchangeably herein to refer to polymers of amino acids of anylength. The polymer may be linear or branched, it may comprise modifiedamino acids, and it may be interrupted by non-amino acids. The termsalso encompass an amino acid polymer that has been modified; forexample, disulfide bond formation, glycosylation, lipidation,acetylation, phosphorylation, or any other manipulation, such asconjugation with a labeling component. As used herein the term “aminoacid” includes natural and/or unnatural or synthetic amino acids,including glycine and both the D or L optical isomers, and amino acidanalogs and peptidomimetics. As used herein, the term “domain” or“protein domain” refers to a part of a protein sequence that may existand function independently of the rest of the protein chain. Asdescribed in aspects of the invention, sequence identity is related tosequence homology. Homology comparisons may be conducted by eye, or moreusually, with the aid of readily available sequence comparison programs.These commercially available computer programs may calculate percent (%)homology between two or more sequences and may also calculate thesequence identity shared by two or more amino acid or nucleic acidsequences.

As used herein the term “wild type” is a term of the art understood byskilled persons and means the typical form of an organism, strain, geneor characteristic as it occurs in nature as distinguished from mutant orvariant forms. A “wild type” can be a base line.

As used herein the term “variant” should be taken to mean the exhibitionof qualities that have a pattern that deviates from what occurs innature. The terms “non-naturally occurring” or “engineered” are usedinterchangeably and indicate the involvement of the hand of man. Theterms, when referring to nucleic acid molecules or polypeptides meanthat the nucleic acid molecule or the polypeptide is at leastsubstantially free from at least one other component with which they arenaturally associated in nature and as found in nature. In all aspectsand embodiments, whether they include these terms or not, it will beunderstood that, preferably, the may be optional and thus preferablyincluded or not preferably not included. Furthermore, the terms“non-naturally occurring” and “engineered” may be used interchangeablyand so can therefore be used alone or in combination and one or othermay replace mention of both together. In particular, “engineered” ispreferred in place of “non-naturally occurring” or “non-naturallyoccurring and/or engineered.”

Sequence homologies may be generated by any of a number of computerprograms known in the art, for example BLAST or FASTA, etc. A suitablecomputer program for carrying out such an alignment is the GCG WisconsinBestfit package (University of Wisconsin, U.S.A; Devereux et al., 1984,Nucleic Acids Research 12:387). Examples of other software than mayperform sequence comparisons include, but are not limited to, the BLASTpackage (see Ausubel et al., 1999 ibid—Chapter 18), FASTA (Atschul etal., 1990, J. Mol. Biol., 403-410) and the GENEWORKS suite of comparisontools. Both BLAST and FASTA are available for offline and onlinesearching (see Ausubel et al., 1999 ibid, pages 7-58 to 7-60). Howeverit is preferred to use the GCG Bestfit program. Percentage (%) sequencehomology may be calculated over contiguous sequences, i.e., one sequenceis aligned with the other sequence and each amino acid or nucleotide inone sequence is directly compared with the corresponding amino acid ornucleotide in the other sequence, one residue at a time. This is calledan “ungapped” alignment. Typically, such ungapped alignments areperformed only over a relatively short number of residues. Although thisis a very simple and consistent method, it fails to take intoconsideration that, for example, in an otherwise identical pair ofsequences, one insertion or deletion may cause the following amino acidresidues to be put out of alignment, thus potentially resulting in alarge reduction in % homology when a global alignment is performed.Consequently, most sequence comparison methods are designed to produceoptimal alignments that take into consideration possible insertions anddeletions without unduly penalizing the overall homology or identityscore. This is achieved by inserting “gaps” in the sequence alignment totry to maximize local homology or identity. However, these more complexmethods assign “gap penalties” to each gap that occurs in the alignmentso that, for the same number of identical amino acids, a sequencealignment with as few gaps as possible —reflecting higher relatednessbetween the two compared sequences—may achieve a higher score than onewith many gaps. “Affinity gap costs” are typically used that charge arelatively high cost for the existence of a gap and a smaller penaltyfor each subsequent residue in the gap. This is the most commonly usedgap scoring system. High gap penalties may, of course, produce optimizedalignments with fewer gaps. Most alignment programs allow the gappenalties to be modified. However, it is preferred to use the defaultvalues when using such software for sequence comparisons. For example,when using the GCG Wisconsin Bestfit package the default gap penalty foramino acid sequences is −12 for a gap and −4 for each extension.Calculation of maximum % homology therefore first requires theproduction of an optimal alignment, taking into consideration gappenalties. A suitable computer program for carrying out such analignment is the GCG Wisconsin Bestfit package (Devereux et al., 1984Nuc. Acids Research 12 p387). Examples of other software than mayperform sequence comparisons include, but are not limited to, the BLASTpackage (see Ausubel et al., 1999 Short Protocols in Molecular Biology,4^(th) Ed. —Chapter 18), FASTA (Altschul et al., 1990 J. Mol. Biol.403-410) and the GENEWORKS suite of comparison tools. Both BLAST andFASTA are available for offline and online searching (see Ausubel etal., 1999, Short Protocols in Molecular Biology, pages 7-58 to 7-60).However, for some applications, it is preferred to use the GCG Bestfitprogram. A new tool, called BLAST 2 Sequences is also available forcomparing protein and nucleotide sequences (see FEMS Microbiol Lett.1999 174(2): 247-50; FEMS Microbiol Lett. 1999 177(1): 187-8 and the website of the National Center for Biotechnology information at the website of the National Institutes for Health). Although the final %homology may be measured in terms of identity, the alignment processitself is typically not based on an all-or-nothing pair comparison.Instead, a scaled similarity score matrix is generally used that assignsscores to each pair-wise comparison based on chemical similarity orevolutionary distance. An example of such a matrix commonly used is theBLOSUM62 matrix—the default matrix for the BLAST suite of programs. GCGWisconsin programs generally use either the public default values or acustom symbol comparison table, if supplied (see user manual for furtherdetails). For some applications, it is preferred to use the publicdefault values for the GCG package, or in the case of other software,the default matrix, such as BLOSUM62. Alternatively, percentagehomologies may be calculated using the multiple alignment feature inDNASIS™ (Hitachi Software), based on an algorithm, analogous to CLUSTAL(Higgins D G & Sharp P M (1988), Gene 73(1), 237-244). Once the softwarehas produced an optimal alignment, it is possible to calculate %homology, preferably % sequence identity. The software typically doesthis as part of the sequence comparison and generates a numericalresult. The sequences may also have deletions, insertions orsubstitutions of amino acid residues which produce a silent change andresult in a functionally equivalent substance. Deliberate amino acidsubstitutions may be made on the basis of similarity in amino acidproperties (such as polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues) and it istherefore useful to group amino acids together in functional groups.Amino acids may be grouped together based on the properties of theirside chains alone. However, it is more useful to include mutation dataas well. The sets of amino acids thus derived are likely to be conservedfor structural reasons. These sets may be described in the form of aVenn diagram (Livingstone C. D. and Barton G. J. (1993) “Proteinsequence alignments: a strategy for the hierarchical analysis of residueconservation” Comput. Appl. Biosci. 9: 745-756) (Taylor W. R. (1986)“The classification of amino acid conservation” J. Theor. Biol. 119;205-218). Conservative substitutions (also with reference to discussionof same herein in conjunction with Table 1A or Table 1B) may be made,for example according to the table below which describes a generallyaccepted Venn diagram grouping of amino acids.

Set Sub-set Hydrophobic F W Y H K M I LV A G C Aromatic F W Y H(SEQ ID NO: 17) (SEQ ID NO: 20) Aliphatic I LV PolarW Y H K R E D C S T N Q Charged H K R E D (SEQ ID NO: 18)(SEQ ID NO: 21) Positively H K R charged Negatively E D charged SmallV C A G S P T N D Tiny A G S (SEQ ID NO: 19)The citations herein concerning conservative substitutions inconjunction with Table 1A or Table 1B are also mentioned to refer tosame as to conservative substitutions.

The terms “subject,” “individual,” and “patient” are usedinterchangeably herein to refer to a vertebrate, preferably a mammal,more preferably a human. Mammals include, but are not limited to,murines, simians, humans, farm animals, sport animals, and pets.Tissues, cells and their progeny of a biological entity obtained in vivoor cultured in vitro are also encompassed.

The terms “therapeutic agent”, “therapeutic capable agent” or “treatmentagent” are used interchangeably and refer to a molecule or compound thatconfers some beneficial effect upon administration to a subject. Thebeneficial effect includes enablement of diagnostic determinations;amelioration of a disease, symptom, disorder, or pathological condition;reducing or preventing the onset of a disease, symptom, disorder orcondition; and generally counteracting a disease, symptom, disorder orpathological condition. As used herein, “treatment” or “treating,” or“palliating” or “ameliorating” are used interchangeably. These termsrefer to an approach for obtaining beneficial or desired resultsincluding but not limited to a therapeutic benefit and/or a prophylacticbenefit. By therapeutic benefit is meant any therapeutically relevantimprovement in or effect on one or more diseases, conditions, orsymptoms under treatment. For prophylactic benefit, the compositions maybe administered to a subject at risk of developing a particular disease,condition, or symptom, or to a subject reporting one or more of thephysiological symptoms of a disease, even though the disease, condition,or symptom may not have yet been manifested. The term “effective amount”or “therapeutically effective amount” refers to the amount of an agentthat is sufficient to effect beneficial or desired results. Thetherapeutically effective amount may vary depending upon one or more of:the subject and disease condition being treated, the weight and age ofthe subject, the severity of the disease condition, the manner ofadministration and the like, which can readily be determined by one ofordinary skill in the art. The term also applies to a dose that willprovide an image for detection by any one of the imaging methodsdescribed herein. The specific dose may vary depending on one or moreof: the particular agent chosen, the dosing regimen to be followed,whether it is administered in combination with other compounds, timingof administration, the tissue to be imaged, and the physical deliverysystem in which it is carried.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of immunology, biochemistry,chemistry, molecular biology, microbiology, cell biology, genomics andrecombinant DNA, which are within the skill of the art. See Sambrook,Fritsch and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2ndedition (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel,et al. eds., (1987)); the series METHODS IN ENZYMOLOGY (Academic Press,Inc.): PCR 2: A PRACTICAL APPROACH (M. J. MacPherson, B. D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, ALABORATORY MANUAL, and ANIMAL CELL CULTURE (R. I. Freshney, ed. (1987)).Several aspects of the invention relate to vector systems comprising oneor more vectors, or vectors as such. Vectors can be designed forexpression of CRISPR transcripts (e.g. nucleic acid transcripts,proteins, or enzymes) in prokaryotic or eukaryotic cells. For example,CRISPR transcripts can be expressed in bacterial cells such asEscherichia coli, insect cells (using baculovirus expression vectors),yeast cells, or mammalian cells. Suitable host cells are discussedfurther in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY185, Academic Press, San Diego, Calif. (1990). Alternatively, therecombinant expression vector can be transcribed and translated invitro, for example using T7 promoter regulatory sequences and T7polymerase. Embodiments of the invention include sequences (bothpolynucleotide or polypeptide) which may comprise homologoussubstitution (substitution and replacement are both used herein to meanthe interchange of an existing amino acid residue or nucleotide, with analternative residue or nucleotide) that may occur i.e., like-for-likesubstitution in the case of amino acids such as basic for basic, acidicfor acidic, polar for polar, etc. Non-homologous substitution may alsooccur i.e., from one class of residue to another or alternativelyinvolving the inclusion of unnatural amino acids such as ornithine(hereinafter referred to as Z), diaminobutyric acid ornithine(hereinafter referred to as B), norleucine ornithine (hereinafterreferred to as 0), pyridylalanine, thienylalanine, naphthylalanine andphenylglycine. Variant amino acid sequences may include suitable spacergroups that may be inserted between any two amino acid residues of thesequence including alkyl groups such as methyl, ethyl or propyl groupsin addition to amino acid spacers such as glycine or β-alanine residues.A further form of variation, which involves the presence of one or moreamino acid residues in peptoid form, may be well understood by thoseskilled in the art. For the avoidance of doubt, “the peptoid form” isused to refer to variant amino acid residues wherein the α-carbonsubstituent group is on the residue's nitrogen atom rather than theα-carbon. Processes for preparing peptides in the peptoid form are knownin the art, for example Simon R J et al., PNAS (1992) 89(20), 9367-9371and Horwell D C, Trends Biotechnol. (1995) 13(4), 132-134. Homologymodelling: Corresponding residues in other Cas13b orthologs can beidentified by the methods of Zhang et al., 2012 (Nature; 490(7421):556-60) and Chen et al., 2015 (PLoS Comput Biol; 11(5): e1004248)—acomputational protein-protein interaction (PPI) method to predictinteractions mediated by domain-motif interfaces. PrePPI (PredictingPPI), a structure based PPI prediction method, combines structuralevidence with non-structural evidence using a Bayesian statisticalframework. The method involves taking a pair a query proteins and usingstructural alignment to identify structural representatives thatcorrespond to either their experimentally determined structures orhomology models. Structural alignment is further used to identify bothclose and remote structural neighbors by considering global and localgeometric relationships. Whenever two neighbors of the structuralrepresentatives form a complex reported in the Protein Data Bank, thisdefines a template for modelling the interaction between the two queryproteins. Models of the complex are created by superimposing therepresentative structures on their corresponding structural neighbor inthe template. This approach is further described in Dey et al., 2013(Prot Sci; 22: 359-66).

For purpose of this invention, amplification means any method employinga primer and a polymerase capable of replicating a target sequence withreasonable fidelity. Amplification may be carried out by natural orrecombinant DNA polymerases such as TagGold™, T7 DNA polymerase, Klenowfragment of E. coli DNA polymerase, and reverse transcriptase. Apreferred amplification method is PCR. In certain aspects the inventioninvolves vectors. A used herein, a “vector” is a tool that allows orfacilitates the transfer of an entity from one environment to another.It is a replicon, such as a plasmid, phage, or cosmid, into whichanother DNA segment may be inserted so as to bring about the replicationof the inserted segment. Generally, a vector is capable of replicationwhen associated with the proper control elements. In general, the term“vector” refers to a nucleic acid molecule capable of transportinganother nucleic acid to which it has been linked. Vectors include, butare not limited to, nucleic acid molecules that are single-stranded,double-stranded, or partially double-stranded; nucleic acid moleculesthat comprise one or more free ends, no free ends (e.g., circular);nucleic acid molecules that comprise DNA, RNA, or both; and othervarieties of polynucleotides known in the art. One type of vector is a“plasmid,” which refers to a circular double stranded DNA loop intowhich additional DNA segments can be inserted, such as by standardmolecular cloning techniques. Another type of vector is a viral vector,wherein virally-derived DNA or RNA sequences are present in the vectorfor packaging into a virus (e.g., retroviruses, replication defectiveretroviruses, adenoviruses, replication defective adenoviruses, andadeno-associated viruses (AAVs)). Viral vectors also includepolynucleotides carried by a virus for transfection into a host cell.Certain vectors are capable of autonomous replication in a host cellinto which they are introduced (e.g., bacterial vectors having abacterial origin of replication and episomal mammalian vectors). Othervectors (e.g., non-episomal mammalian vectors) are integrated into thegenome of a host cell upon introduction into the host cell, and therebyare replicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively-linked. Such vectors are referred to herein as “expressionvectors.” Common expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. Recombinant expressionvectors can comprise a nucleic acid of the invention in a form suitablefor expression of the nucleic acid in a host cell, which means that therecombinant expression vectors include one or more regulatory elements,which may be selected on the basis of the host cells to be used forexpression, that is operatively-linked to the nucleic acid sequence tobe expressed. Within a recombinant expression vector, “operably linked”is intended to mean that the nucleotide sequence of interest is linkedto the regulatory element(s) in a manner that allows for expression ofthe nucleotide sequence (e.g., in an in vitro transcription/translationsystem or in a host cell when the vector is introduced into the hostcell). With regards to recombination and cloning methods, mention ismade of U.S. patent application Ser. No. 10/815,730, published Sep. 2,2004 as US 2004-0171156 A1, the contents of which are hereinincorporated by reference in their entirety. Aspects of the inventionrelate to bicistronic vectors for guide RNA and wild type, modified ormutated CRISPR effector proteins/enzymes (e.g. Cas13b effectorproteins). Bicistronic expression vectors guide RNA and wild type,modified or mutated CRISPR effector proteins/enzymes (e.g. Cas13beffector proteins) are preferred. In general and particularly in thisembodiment and wild type, modified or mutated CRISPR effectorproteins/enzymes (e.g. Cas13b effector proteins) is preferably driven bythe CBh promoter. The RNA may preferably be driven by a Pol IIIpromoter, such as a U6 promoter. Ideally the two are combined.

In some embodiments, a loop in the guide RNA or crRNA is provided. Thismay be a stem loop or a tetra loop. The loop is preferably GAAA, but itis not limited to this sequence or indeed to being only 4 bp in length.Indeed, preferred loop forming sequences for use in hairpin structuresare four nucleotides in length, and most preferably have the sequenceGAAA. However, longer or shorter loop sequences may be used, as mayalternative sequences. The sequences preferably include a nucleotidetriplet (for example, AAA), and an additional nucleotide (for example Cor G). Examples of loop forming sequences include CAAA and AAAG.

In practicing any of the methods disclosed herein, a suitable vector canbe introduced to a cell or an embryo via one or more methods known inthe art, including without limitation, microinjection, electroporation,sonoporation, biolistics, calcium phosphate-mediated transfection,cationic transfection, liposome transfection, dendrimer transfection,heat shock transfection, nucleofection transfection, magnetofection,lipofection, impalefection, optical transfection, proprietaryagent-enhanced uptake of nucleic acids, and delivery via liposomes,immunoliposomes, virosomes, or artificial virions. In some methods, thevector is introduced into an embryo by microinjection. The vector orvectors may be microinjected into the nucleus or the cytoplasm of theembryo. In some methods, the vector or vectors may be introduced into acell by nucleofection.

Vectors can be designed for expression of CRISPR transcripts (e.g.,nucleic acid transcripts, proteins, or enzymes) in prokaryotic oreukaryotic cells. For example, CRISPR transcripts can be expressed inbacterial cells such as Escherichia coli, insect cells (usingbaculovirus expression vectors), yeast cells, or mammalian cells.Suitable host cells are discussed further in Goeddel, GENE EXPRESSIONTECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif(1990). Alternatively, the recombinant expression vector can betranscribed and translated in vitro, for example using T7 promoterregulatory sequences and T7 polymerase.

Vectors may be introduced and propagated in a prokaryote or prokaryoticcell. In some embodiments, a prokaryote is used to amplify copies of avector to be introduced into a eukaryotic cell or as an intermediatevector in the production of a vector to be introduced into a eukaryoticcell (e.g., amplifying a plasmid as part of a viral vector packagingsystem). In some embodiments, a prokaryote is used to amplify copies ofa vector and express one or more nucleic acids, such as to provide asource of one or more proteins for delivery to a host cell or hostorganism. Expression of proteins in prokaryotes is most often carriedout in Escherichia coli with vectors containing constitutive orinducible promoters directing the expression of either fusion ornon-fusion proteins. Fusion vectors add a number of amino acids to aprotein encoded therein, such as to the amino terminus of therecombinant protein. Such fusion vectors may serve one or more purposes,such as: (i) to increase expression of recombinant protein; (ii) toincrease the solubility of the recombinant protein; and (iii) to aid inthe purification of the recombinant protein by acting as a ligand inaffinity purification. Often, in fusion expression vectors, aproteolytic cleavage site is introduced at the junction of the fusionmoiety and the recombinant protein to enable separation of therecombinant protein from the fusion moiety subsequent to purification ofthe fusion protein. Such enzymes, and their cognate recognitionsequences, include Factor Xa, thrombin and enterokinase. Example fusionexpression vectors include pGEX (Pharmacia Biotech Inc; Smith andJohnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly,Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuse glutathioneS-transferase (GST), maltose E binding protein, or protein A,respectively, to the target recombinant protein. Examples of suitableinducible non-fusion E. coli expression vectors include pTrc (Amrann etal., (1988) Gene 69:301 315) and pET 11d (Studier et al., GENEEXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, SanDiego, Calif. (1990) 60-89). In some embodiments, a vector is a yeastexpression vector. Examples of vectors for expression in yeastSaccharomyces cerevisiae include pYepSec1 (Baldari, et al., 1987. EMBOJ. 6: 229-234), pMFa (Kuijan and Herskowitz, 1982. Cell 30: 933-943),pJRY88 (Schultz et al., 1987. Gene 54: 113-123), pYES2 (InvitrogenCorporation, San Diego, Calif.), and picZ (InVitrogen Corp, San Diego,Calif). In some embodiments, a vector drives protein expression ininsect cells using baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., SF9cells) include the pAc series (Smith, et al., 1983. Mol. Cell. Biol. 3:2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology 170:31-39). In some embodiments, a vector is capable of driving expressionof one or more sequences in mammalian cells using a mammalian expressionvector. Examples of mammalian expression vectors include pCDM8 (Seed,1987. Nature 329: 840) and pMT2PC (Kaufman, et al., 1987. EMBO J. 6:187-195). When used in mammalian cells, the expression vector's controlfunctions are typically provided by one or more regulatory elements. Forexample, commonly used promoters are derived from polyoma, adenovirus 2,cytomegalovirus, simian virus 40, and others disclosed herein and knownin the art. For other suitable expression systems for both prokaryoticand eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al.,MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989. In some embodiments, the recombinant mammalian expressionvector is capable of directing expression of the nucleic acidpreferentially in a particular cell type (e.g., tissue-specificregulatory elements are used to express the nucleic acid).Tissue-specific regulatory elements are known in the art. Non-limitingexamples of suitable tissue-specific promoters include the albuminpromoter (liver-specific; Pinkert, et al., 1987. Genes Dev. 1: 268-277),lymphoid-specific promoters (Calame and Eaton, 1988. Adv. Immunol. 43:235-275), in particular promoters of T cell receptors (Winoto andBaltimore, 1989. EMBO J. 8: 729-733) and immunoglobulins (Baneiji, etal., 1983. Cell 33: 729-740; Queen and Baltimore, 1983. Cell 33:741-748), neuron-specific promoters (e.g., the neurofilament promoter;Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci. USA 86: 5473-5477),pancreas-specific promoters (Edlund, et al., 1985. Science 230:912-916), and mammary gland-specific promoters (e.g., milk wheypromoter; U.S. Pat. No. 4,873,316 and European Application PublicationNo. 264,166). Developmentally-regulated promoters are also encompassed,e.g., the murine hox promoters (Kessel and Gruss, 1990. Science 249:374-379) and the α-fetoprotein promoter (Campes and Tilghman, 1989.Genes Dev. 3: 537-546). With regards to these prokaryotic and eukaryoticvectors, mention is made of U.S. Pat. No. 6,750,059, the contents ofwhich are incorporated by reference herein in their entirety. Otherembodiments of the invention may relate to the use of viral vectors,with regards to which mention is made of U.S. patent application Ser.No. 13/092,085, the contents of which are incorporated by referenceherein in their entirety. Tissue-specific regulatory elements are knownin the art and in this regard, mention is made of U.S. Pat. No.7,776,321, the contents of which are incorporated by reference herein intheir entirety.

In some embodiments, a regulatory element is operably linked to one ormore elements of or encoding a CRISPR Cas13b system or complex so as todrive expression of the one or more elements of the CRISPR system. Ingeneral, CRISPRs (Clustered Regularly Interspaced Short PalindromicRepeats), also known as SPIDRs (SPacer Interspersed Direct Repeats),constitute a family of DNA loci that are usually specific to aparticular bacterial species. The CRISPR locus comprises a distinctclass of interspersed short sequence repeats (SSRs) that were recognizedin E. coli (Ishino et al., J. Bacteriol., 169:5429-5433 [1987]; andNakata et al., J. Bacteriol., 171:3553 3556 [1989]), and associatedgenes. Similar interspersed SSRs have been identified in Haloferaxmediterranei, Streptococcus pyogenes, Anabaena, and Mycobacteriumtuberculosis (See, Groenen et al., Mol. Microbiol., 10:1057-1065 [1993];Hoe et al., Emerg. Infect. Dis., 5:254-263 [1999]; Masepohl et al.,Biochim. Biophys. Acta 1307:26-30 [1996]; and Mojica et al., Mol.Microbiol., 17:85-93 [1995]). The CRISPR loci typically differ fromother SSRs by the structure of the repeats, which have been termed shortregularly spaced repeats (SRSRs) (Janssen et al., OMICS J. Integ. Biol.,6:23-33 [2002]; and Mojica et al., Mol. Microbiol., 36:244-246 [2000]).In general, the repeats are short elements that occur in clusters thatare regularly spaced by unique intervening sequences with asubstantially constant length (Mojica et al., [2000], supra). Althoughthe repeat sequences are highly conserved between strains, the number ofinterspersed repeats and the sequences of the spacer regions typicallydiffer from strain to strain (van Embden et al., J. Bacteriol.,182:2393-2401 [2000]). CRISPR loci have been identified in more than 40prokaryotes (See e.g., Jansen et al., Mol. Microbiol., 43:1565-1575[2002]; and Mojica et al., [2005]) including, but not limited toAeropyrum, Pyrobaculum, Sulfolobus, Archaeoglobus, Haloarcula,Methanobacterium, Methanococcus, Methanosarcina, Methanopyrus,Pyrococcus, Picrophilus, Thermoplasma, Corynebacterium, Mycobacterium,Streptomyces, Aquifex, Porphyromonas, Chlorobium, Thermus, Bacillus,Listeria, Staphylococcus, Clostridium, Thermoanaerobacter, Mycoplasma,Fusobacterium, Azoarcus, Chromobacterium, Neisseria, Nitrosomonas,Desulfovibrio, Geobacter, Myxococcus, Campylobacter, Wolinella,Acinetobacter, Envinia, Escherichia, Legionella, Methylococcus,Pasteurella, Photobacterium, Salmonella, Xanthomonas, Yersinia,Treponema, and Thermotoga.

In general, “RNA-targeting system” as used in the present applicationrefers collectively to transcripts and other elements involved in theexpression of or directing the activity of RNA-targetingCRISPR-associated 13b (“Cas13b”) genes (also referred to herein as aneffector protein), including sequences encoding a RNA-targeting Cas(effector) protein and a guide RNA (or crRNA sequence), with referenceto Table 1A or Table 1B as herein discussed. In general, a RNA-targetingsystem is characterized by elements that promote the formation of aRNA-targeting complex at the site of a target sequence. In the contextof formation of a RNA-targeting complex, “target sequence” refers to aRNA sequence to which a guide sequence (or the guide or of the crRNA) isdesigned to have complementarity, where hybridization between a targetsequence and a guide RNA promotes the formation of a RNA-targetingcomplex. Full complementarity is not necessarily required, providedthere is sufficient complementarity to cause hybridization and promoteformation of a RNA-targeting complex. In some embodiments, a targetsequence is located in the nucleus or cytoplasm of a cell. In someembodiments, the target sequence may be within an organelle of aeukaryotic cell. A sequence or template that may be used forrecombination into the targeted locus comprising the target sequences isreferred to as an “editing template” or “editing RNA” or “editingsequence”. In aspects of the invention, an exogenous template RNA may bereferred to as an editing template. In an aspect of the invention therecombination is homologous recombination. In general, a guide sequenceis any polynucleotide sequence having sufficient complementarity with atarget polynucleotide sequence to hybridize with the target sequence anddirect sequence-specific binding of a nucleic acid-targeting complex tothe target sequence. In some embodiments, the degree of complementaritybetween a guide sequence and its corresponding target sequence, whenoptimally aligned using a suitable alignment algorithm, is about or morethan about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more.Optimal alignment may be determined with the use of any suitablealgorithm for aligning sequences, non-limiting example of which includethe Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithmsbased on the Burrows-Wheeler Transform (e.g. the Burrows WheelerAligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies,ELAND (Illumina, San Diego, CA), SOAP (available atsoap.genomics.org.cn), and Maq (available at maq.sourceforge.net). Insome embodiments, a guide sequence is about or more than about 5, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 35, 40, 45, 50, 75, or more nucleotides in length. In someembodiments, a guide sequence is less than about 75, 50, 45, 40, 35, 30,25, 20, 15, 12, or fewer nucleotides in length. The ability of a guidesequence to direct sequence-specific binding of a RNA-targeting complexto a target sequence may be assessed by any suitable assay. A templatepolynucleotide may be of any suitable length, such as about or more thanabout 10, 15, 20, 25, 50, 75, 100, 150, 200, 500, 1000, or morenucleotides in length. In some embodiments, the template polynucleotideis complementary to a portion of a polynucleotide comprising the targetsequence. When optimally aligned, a template polynucleotide mightoverlap with one or more nucleotides of a target sequences (e.g. aboutor more than about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80,90, 100 or more nucleotides). In some embodiments, when a templatesequence and a polynucleotide comprising a target sequence are optimallyaligned, the nearest nucleotide of the template polynucleotide is withinabout 1, 5, 10, 15, 20, 25, 50, 75, 100, 200, 300, 400, 500, 1000, 5000,10000, or more nucleotides from the target sequence. In someembodiments, the RNA-targeting effector protein is part of a fusionprotein comprising one or more heterologous protein domains (e.g., aboutor more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more domains inaddition to the nucleic acid-targeting effector protein). In someembodiments, the CRISPR Cas13b effector protein/enzyme is part of afusion protein comprising one or more heterologous protein domains (e.g.about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more domainsin addition to the CRISPR Cas13b enzyme). Examples of protein domainsthat may be fused to an effector protein include, without limitation,epitope tags, reporter gene sequences, and protein domains having one ormore of the following activities: methylase activity, demethylaseactivity, transcription activation activity, transcription repressionactivity, transcription release factor activity, histone modificationactivity, RNA cleavage activity and nucleic acid binding activity.Non-limiting examples of epitope tags include histidine (His) tags, V5tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-Gtags, and thioredoxin (Trx) tags. Examples of reporter genes include,but are not limited to, glutathione-S-transferase (GST), horseradishperoxidase (HRP), chloramphenicol acetyltransferase (CAT)beta-galactosidase, beta-glucuronidase, luciferase, green fluorescentprotein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellowfluorescent protein (YFP), and autofluorescent proteins including bluefluorescent protein (BFP). A nucleic acid-targeting effector protein maybe fused to a gene sequence encoding a protein or a fragment of aprotein that bind DNA molecules or bind other cellular molecules,including but not limited to maltose binding protein (MBP), S-tag, Lex ADNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, andherpes simplex virus (HSV) BP16 protein fusions. Additional domains thatmay form part of a fusion protein comprising a nucleic acid-targetingeffector protein are described in US20110059502, incorporated herein byreference. In some embodiments, a tagged nucleic acid-targeting effectorprotein is used to identify the location of a target sequence. In someembodiments, a CRISPR Cas13b enzyme may form a component of an induciblesystem. The inducible nature of the system would allow forspatiotemporal control of gene editing or gene expression using a formof energy. The form of energy may include but is not limited toelectromagnetic radiation, sound energy, chemical energy and thermalenergy. Examples of inducible system include tetracycline induciblepromoters (Tet-On or Tet-Off), small molecule two-hybrid transcriptionactivations systems (FKBP, ABA, etc), or light inducible systems(Phytochrome, LOV domains, or cryptochrome). In one embodiment, theCRISPR Cas13b enzyme may be a part of a Light Inducible TranscriptionalEffector (LITE) to direct changes in transcriptional activity in asequence-specific manner. The components of a light may include a CRISPRenzyme, a light-responsive cytochrome heterodimer (e.g. from Arabidopsisthaliana), and a transcriptional activation/repression domain. Furtherexamples of inducible DNA binding proteins and methods for their use areprovided in U.S. 61/736,465 and U.S. 61/721,283 and WO 2014/018423 andU.S. Pat. Nos. 8,889,418, 8,895,308, US20140186919, US20140242700,US20140273234, US20140335620, WO 2014/093635, which is herebyincorporated by reference in its entirety. In some aspects, theinvention provides methods comprising delivering one or morepolynucleotides, such as or one or more vectors as described herein, oneor more transcripts thereof, and/or one or proteins transcribedtherefrom, to a host cell. In some aspects, the invention furtherprovides cells produced by such methods, and organisms (such as animals,plants, or fungi) comprising or produced from such cells. In someembodiments, a RNA-targeting effector protein in combination with (andoptionally complexed with) a guide RNA or crRNA is delivered to a cell.Conventional viral and non-viral based gene transfer methods can be usedto introduce nucleic acids in mammalian cells or target tissues. Suchmethods can be used to administer nucleic acids encoding components of aRNA-targeting system to cells in culture, or in a host organism.Non-viral vector delivery systems include DNA plasmids, RNA (e.g. atranscript of a vector described herein), naked nucleic acid, andnucleic acid complexed with a delivery vehicle, such as a liposome.Viral vector delivery systems include DNA and RNA viruses, which haveeither episomal or integrated genomes after delivery to the cell. For areview of gene therapy procedures, see Anderson, Science 256:808-813(1992); Nabel & Felgner, TIBTECH 11:211-217 (1993); Mitani & Caskey,TIBTECH 11:162-166 (1993); Dillon, TIBTECH 11:167-175 (1993); Miller,Nature 357:455-460 (1992); Van Brunt, Biotechnology 6(10):1149-1154(1988); Vigne, Restorative Neurology and Neuroscience 8:35-36 (1995);Kremer & Perricaudet, British Medical Bulletin 51(1):31-44 (1995);Haddada et al., in Current Topics in Microbiology and Immunology,Doerfler and Böhm (eds) (1995); and Yu et al., Gene Therapy 1:13-26(1994). Methods of non-viral delivery of nucleic acids includelipofection, nucleofection, microinjection, biolistics, virosomes,liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates,naked DNA, artificial virions, and agent-enhanced uptake of DNA.Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787;and 4,897,355) and lipofection reagents are sold commercially (e.g.,Transfectam™ and Lipofectin™). Cationic and neutral lipids that aresuitable for efficient receptor-recognition lipofection ofpolynucleotides include those of Felgner, WO 91/17424; WO 91/16024.Delivery can be to cells (e.g. in vitro or ex vivo administration) ortarget tissues (e.g. in vivo administration).

Models of Conditions

A method of the invention may be used to create a plant, an animal orcell that may be used to model and/or study genetic or epigeneticconditions of interest, such as a through a model of mutations ofinterest or a disease model. As used herein, “disease” refers to adisease, disorder, or indication in a subject. For example, a method ofthe invention may be used to create an animal or cell that comprises amodification in one or more nucleic acid sequences associated with adisease, or a plant, animal or cell in which expression of one or morenucleic acid sequences associated with a disease are altered. Such anucleic acid sequence may encode or be translated a disease associatedprotein sequence or may be a disease associated control sequence.Accordingly, it is understood that in embodiments of the invention, aplant, subject, patient, organism or cell can be a non-human subject,patient, organism or cell. Thus, the invention provides a plant, animalor cell, produced by the present methods, or a progeny thereof. Theprogeny may be a clone of the produced plant or animal, or may resultfrom sexual reproduction by crossing with other individuals of the samespecies to introgress further desirable traits into their offspring. Thecell may be in vivo or ex vivo in the cases of multicellular organisms,particularly animals or plants. In the instance where the cell is incultured, a cell line may be established if appropriate culturingconditions are met and preferably if the cell is suitably adapted forthis purpose (for instance a stem cell). Bacterial cell lines producedby the invention are also envisaged. Hence, cell lines are alsoenvisaged. In some methods, the disease model can be used to study theeffects of mutations, or more general altered, such as reduced,expression of genes or gene products on the animal or cell anddevelopment and/or progression of the disease using measures commonlyused in the study of the disease. Alternatively, such a disease model isuseful for studying the effect of a pharmaceutically active compound onthe disease. In some methods, the disease model can be used to assessthe efficacy of a potential gene therapy strategy. That is, adisease-associated RNA can be modified such that the disease developmentand/or progression is displayed or inhibited or reduced and then effectsof a compound on the progression or inhibition or reduction are tested.

Useful in the practice of the instant invention utilizing Table 1A orTable 1B Cas13b effector proteins and complexes thereof and nucleic acidmolecules encoding same and methods using same, reference is made to:Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells. Shalem, O.,Sanjana, N E., Hartenian, E., Shi, X., Scott, D A., Mikkelson, T.,Heckl, D., Ebert, B L., Root, D E., Doench, J G., Zhang, F. Science Dec.12. (2013). [Epub ahead of print]; Published in final edited form as:Science. 2014 Jan. 3; 343(6166): 84-87. Shalem et al. involves a new wayto interrogate gene function on a genome-wide scale. Their studiesshowed that delivery of a genome-scale CRISPR-Cas9 knockout (GeCKO)library targeted 18,080 genes with 64,751 unique guide sequences enabledboth negative and positive selection screening in human cells. First,the authors showed use of the GeCKO library to identify genes essentialfor cell viability in cancer and pluripotent stem cells. Next, in amelanoma model, the authors screened for genes whose loss is involved inresistance to vemurafenib, a therapeutic that inhibits mutant proteinkinase BRAF. Their studies showed that the highest-ranking candidatesincluded previously validated genes NF1 and MED12 as well as novelhitsNF2, CUL3, TADA2B, and TADA1. The authors observed a high level ofconsistency between independent guide RNAs targeting the same gene and ahigh rate of hit confirmation, and thus demonstrated the promise ofgenome-scale screening with Cas9. Reference is also made to US patentpublication number US20140357530; and PCT Patent Publication WO2014/093701, hereby incorporated herein by reference.

The term “associated with” is used here in relation to the associationof the functional domain to the Cas13b effector protein or the adaptorprotein. It is used in respect of how one molecule associates' withrespect to another, for example between an adaptor protein and afunctional domain, or between the Cas13b effector protein and afunctional domain. In the case of such protein-protein interactions,this association may be viewed in terms of recognition in the way anantibody recognizes an epitope. Alternatively, one protein may beassociated with another protein via a fusion of the two, for instanceone subunit being fused to another subunit. Fusion typically occurs byaddition of the amino acid sequence of one to that of the other, forinstance via splicing together of the nucleotide sequences that encodeeach protein or subunit. Alternatively, this may essentially be viewedas binding between two molecules or direct linkage, such as a fusionprotein. In any event, the fusion protein may include a linker betweenthe two subunits of interest (i.e. between the enzyme and the functionaldomain or between the adaptor protein and the functional domain). Thus,in some embodiments, the Cas13b effector protein or adaptor protein isassociated with a functional domain by binding thereto. In otherembodiments, the Cas13b effector protein or adaptor protein isassociated with a functional domain because the two are fused together,optionally via an intermediate linker.

Cas13b Effector Protein Complexes can be Used in Plants

The invention in some embodiments comprehends a method of modifying ancell or organism. The cell may be a prokaryotic cell or a eukaryoticcell. The cell may be a mammalian cell. The mammalian cell many be anon-human primate, bovine, porcine, rodent or mouse cell. The cell maybe a non-mammalian eukaryotic cell such as poultry, fish or shrimp. Thecell may also be a plant cell. The plant cell may be of a crop plantsuch as cassava, corn, sorghum, wheat, or rice. The plant cell may alsobe of an algae, tree or vegetable. The modification introduced to thecell by the present invention may be such that the cell and progeny ofthe cell are altered for improved production of biologic products suchas an antibody, starch, alcohol or other desired cellular output. Themodification introduced to the cell by the present invention may be suchthat the cell and progeny of the cell include an alteration that changesthe biologic product produced. The system may comprise one or moredifferent vectors. In an aspect of the invention, the effector proteinis codon optimized for expression the desired cell type, preferentiallya eukaryotic cell, preferably a mammalian cell or a human cell. Cas13bsystem(s) (e.g., single or multiplexed) can be used in conjunction withrecent advances in crop genomics. Such CRISPR system(s) can be used toperform efficient and cost effective plant gene or genome ortranscriptome interrogation or editing or manipulation—for instance, forrapid investigation and/or selection and/or interrogations and/orcomparison and/or manipulations and/or transformation of plant genes orgenomes; e.g., to create, identify, develop, optimize, or confertrait(s) or characteristic(s) to plant(s) or to transform a plantgenome. There can accordingly be improved production of plants, newplants with new combinations of traits or characteristics or new plantswith enhanced traits. Such CRISPR system(s) can be used with regard toplants in Site-Directed Integration (SDI) or Gene Editing (GE) or anyNear Reverse Breeding (NRB) or Reverse Breeding (RB) techniques.Accordingly, reference herein to animal cells may also apply, mutatismutandis, to plant cells unless otherwise apparent; and, the enzymesherein having reduced off-target effects and systems employing suchenzymes can be used in plant applications, including those mentionedherein. Engineered plants modified by the effector protein (Table 1A orTable 1B Cas13b) and suitable guide (crRNA), and progeny thereof, asprovided. These may include disease or drought resistant crops, such aswheat, barley, rice, soybean or corn; plants modified to remove orreduce the ability to self-pollinate (but which can instead, optionally,hybridise instead); and allergenic foods such as peanuts and nuts wherethe immunogenic proteins have been disabled, destroyed or disrupted bytargeting via a effector protein and suitable guide. Any aspect of usingclassical CRISPR-Cas systems may be adapted to use in CRISPR systemsthat are Cas protein agnostic, e.g. Cas13b effector protein systems.

Therapeutic Treatment

The system of the invention can be applied in areas of former RNAcutting technologies, without undue experimentation, from thisdisclosure, including therapeutic, assay and other applications, becausethe present application provides the foundation for informed engineeringof the system. The present invention provides for therapeutic treatmentof a disease caused by overexpression of RNA, toxic RNA and/or mutatedRNA (such as, for example, splicing defects or truncations). Expressionof the toxic RNA may be associated with formation of nuclear inclusionsand late-onset degenerative changes in brain, heart or skeletal muscle.In the best studied example, myotonic dystrophy, it appears that themain pathogenic effect of the toxic RNA is to sequester binding proteinsand compromise the regulation of alternative splicing (Hum. Mol. Genet.(2006) 15 (suppl 2): R162-R169). Myotonic dystrophy [dystrophiamyotonica (DM)] is of particular interest to geneticists because itproduces an extremely wide range of clinical features. A partial listingwould include muscle wasting, cataracts, insulin resistance, testicularatrophy, slowing of cardiac conduction, cutaneous tumors and effects oncognition. The classical form of DM, which is now called DM type 1(DM1), is caused by an expansion of CTG repeats in the 3′-untranslatedregion (UTR) of DMPK, a gene encoding a cytosolic protein kinase.

The innate immune system detects viral infection primarily byrecognizing viral nucleic acids inside an infected cell, referred to asDNA or RNA sensing. In vitro RNA sensing assays can be used to detectspecific RNA substrates. The RNA targeting effector protein can forinstance be used for RNA-based sensing in living cells. Examples ofapplications are diagnostics by sensing of, for examples,disease-specific RNAs. The RNA targeting effector protein (Table 1A orTable 1B Cas13b) of the invention can further be used for antiviralactivity, in particular against RNA viruses. The effector protein (Table1A or Table 1B Cas13b) can be targeted to the viral RNA using a suitableguide RNA selective for a selected viral RNA sequence. In particular,the effector protein may be an active nuclease that cleaves RNA, such assingle stranded RNA. Therapeutic dosages of the enzyme system of thepresent invention to target RNA the above-referenced RNAs arecontemplated to be about 0.1 to about 2 mg/kg the dosages may beadministered sequentially with a monitored response, and repeateddosages if necessary, up to about 7 to 10 doses per patient.Advantageously, samples are collected from each patient during thetreatment regimen to ascertain the effectiveness of treatment. Forexample, RNA samples may be isolated and quantified to determine ifexpression is reduced or ameliorated. Such a diagnostic is within thepurview of one of skill in the art.

Transcript Detection Methods

The effector proteins (Table 1A or Table 1B Cas13b) and systems of theinvention are useful for specific detection of RNAs in a cell or othersample. In the presence of an RNA target of interest, guide-dependentCas13b nuclease activity may be accompanied by non-specific RNAseactivity against collateral targets. To take advantage of the RNaseactivity, all that is needed is a reporter substrate that can bedetectably cleaved. For example, a reporter molecule can comprise RNA,tagged with a fluorescent reporter molecule (fluor) on one end and aquencher on the other. In the absence of Cas13b RNase activity, thephysical proximity of the quencher dampens fluorescence from the fluorto low levels. When Cas13b target specific cleavage is activated by thepresence of an RNA target-of-interest and suitable guide RNA, theRNA-containing reporter molecule is non-specifically cleaved and thefluor and quencher are spatially separated. This causes the fluor toemit a detectable signal when excited by light of the appropriatewavelength. In one exemplary assay method, Cas13b effector,target-of-interest-specific guide RNA, and reporter molecule are addedto a cellular sample. An increase in fluorescence indicates the presenceof the RNA target-of-interest. In another exemplary method, a detectionarray is provided. Each location of the array is provided with Cas13beffector, reporter molecule, and a target-of-interest-specific guideRNA. Depending on the assay to be performed, thetarget-of-interest-specific guide RNAs at each location of the array canbe the same, different, or a combination thereof. Differenttarget-of-interest-specific guide RNAs might be provided, for examplewhen it is desired to test for one or more targets in a single sourcesample. The same target-of-interest-specific guide RNA might be providedat each location, for example when it is desired to test multiplesamples for the same target.

In certain embodiments, Cas13b is provided or expressed in an in vitrosystem or in a cell, transiently or stably, and targeted or triggered tonon-specifically cleave cellular nucleic acids. In one embodiment,Cas13b is engineered to knock down ssDNA, for example viral ssDNA. Inanother embodiment, Cas13b is engineered to knock down RNA. The systemcan be devised such that the knockdown is dependent on a target DNApresent in the cell or in vitro system, or triggered by the addition ofa target nucleic acid to the system or cell.

In an embodiment, the Cas13b system is engineered to non-specificallycleave RNA in a subset of cells distinguishable by the presence of anaberrant DNA sequence, for instance where cleavage of the aberrant DNAmight be incomplete or ineffectual. In one non-limiting example, a DNAtranslocation that is present in a cancer cell and drives celltransformation is targeted. Whereas a subpopulation of cells thatundergoes chromosomal DNA and repair may survive, non specificcollateral ribonuclease activity advantageously leads to cell death ofpotential survivors.

Collateral activity was recently leveraged for a highly sensitive andspecific nucleic acid detection platform termed SHERLOCK that is usefulfor many clinical diagnoses (Gootenberg, J. S. et al. Nucleic aciddetection with CRISPR-Cas13a/C2c2. Science 356, 438-442 (2017)).

According to the invention, engineered Cas13b systems are optimized forRNA endonuclease activity and can be expressed in mammalian cells andtargeted to effectively knock down reporter molecules or transcripts incells.

The collateral effect of engineered Cas13b with isothermal amplificationprovides a CRISPR-based diagnostic providing rapid DNA or RNA detectionwith high sensitivity and single-base mismatch specificity. TheCas13b-based molecular detection platform is used to detect specificstrains of virus, distinguish pathogenic bacteria, genotype human DNA,and identify cell-free tumor DNA mutations. Furthermore, reactionreagents can be lyophilized for cold-chain independence and long-termstorage, and readily reconstituted on paper for field applications.

The ability to rapidly detect nucleic acids with high sensitivity andsingle-base specificity on a portable platform may aid in diseasediagnosis and monitoring, epidemiology, and general laboratory tasks.Although methods exist for detecting nucleic acids, they have trade-offsamong sensitivity, specificity, simplicity, cost, and speed.

Microbial Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR) and CRISPR-associated (CRISPR-Cas) adaptive immune systemscontain programmable endonucleases that can be leveraged forCRISPR-based diagnostics (CRISPR-Dx). Cas13b can be reprogrammed withCRISPR RNAs (crRNAs) to provide a platform for specific DNA sensing.Upon recognition of its DNA target, activated Cas13b engages in“collateral” cleavage of nearby non-targeted nucleic acids (i.e., RNAand/or ssDNA). This crRNA-programmed collateral cleavage activity allowsCas13b to detect the presence of a specific DNA in vivo by triggeringprogrammed cell death or by nonspecific degradation of labeled RNA orssDNA. Here is described an in vitro nucleic acid detection platformwith high sensitivity based on nucleic acid amplification andCas13b-mediated collateral cleavage of a commercial reporter RNA,allowing for real-time detection of the target.

In certain example embodiments, the Cas13b effector protein is from anorganism identified in Table 1A or Table 1B. In certain exampleembodiments, the Cas13b effector protein is from an organism selectedfrom Bergeyella zoohelcum, Prevotella intermedia, Prevotella buccae,Porphyromonas gingivalis, Bacteroides pyogenes, Alistipes sp. ZOR0009,Prevotella sp. MA2016, Riemerella anatipestifer, Prevotella aurantiaca,Prevotella saccharolytica, Myroides odoratimimus CCUG 10230,Capnocytophaga canimorsus, Porphyromonas gulae, Prevotella sp. P5-125,Flavobacterium branchiophilum, Myroides odoratimimus, Flavobacteriumcolumnare, or Porphyromonas sp. COT-052 OH4946. In another embodiment,the one or more guide RNAs are designed to bind to one or more targetRNA sequences that are diagnostic for a disease state.

In certain example embodiments, an RNA-based masking constructsuppresses generation of a detectable positive signal, or the RNA-basedmasking construct suppresses generation of a detectable positive signalby masking the detectable positive signal, or generating a detectablenegative signal instead, or the RNA-based masking construct comprises asilencing RNA that suppresses generation of a gene product encoded by areporting construct, wherein the gene product generates the detectablepositive signal when expressed.

In another example embodiment, the RNA-based masking construct is aribozyme that generates a negative detectable signal, and wherein thepositive detectable signal is generated when the ribozyme isdeactivated. In one example embodiment, the ribozyme converts asubstrate to a first color and wherein the substrate converts to asecond color when the ribozyme is deactivated. In another exampleembodiment, the RNA-based masking agent is an aptamer that sequesters anenzyme, wherein the enzyme generates a detectable signal upon releasefrom the aptamer by acting upon a substrate, or the aptamer sequesters apair of agents that when released from the aptamers combine to generatea detectable signal.

In another example embodiment, the RNA-based masking construct comprisesan RNA oligonucleotide to which are attached a detectable ligandoligonucleotide and a masking component. In certain example embodiments,the detectable ligand is a fluorophore and the masking component is aquencher molecule.

In another aspect, the invention provides a method for detecting targetRNAs in samples, comprising: distributing a sample or set of samplesinto one or more individual discrete volumes, the individual discretevolumes comprising a CRISPR system comprising an effector protein, oneor more guide RNAs, an RNA-based masking construct; incubating thesample or set of samples under conditions sufficient to allow binding ofthe one or more guide RNAs to one or more target molecules; activatingthe CRISPR effector protein via binding of the one or more guide RNAs tothe one or more target molecules, wherein activating the CRISPR effectorprotein results in modification of the RNA-based masking construct suchthat a detectable positive signal is produced; and detecting thedetectable positive signal, wherein detection of the detectable positivesignal indicates a presence of one or more target molecules in thesample.

In another aspect, the invention provides a method for detectingpeptides in samples, comprising: distributing a sample or set of samplesinto a set of individual discrete volumes, the individual discretevolumes comprising peptide detection aptamers, a CRISPR systemcomprising an effector protein, one or more guide RNAs, an RNA-basedmasking construct, wherein the peptide detection aptamers comprising amasked RNA polymerase site and configured to bind one or more targetmolecules; incubating the sample or set of samples under conditionssufficient to allow binding of the peptide detection aptamers to the oneor more target molecules, wherein binding of the aptamer to acorresponding target molecule exposes the RNA polymerase binding siteresulting in RNA synthesis of a trigger RNA; activating the CRISPReffector protein via binding of the one or more guide RNAs to thetrigger RNA, wherein activating the CRISPR effector protein results inmodification of the RNA-based masking construct such that a detectablepositive signal is produced; and detecting the detectable positivesignal, wherein detection of the detectable positive signal indicates apresence of one or more target molecules in a sample.

In certain example embodiments, the one or more guide RNAs are designedto bind to one or more target molecules that are diagnostic for adisease state. In certain other example embodiments, the disease stateis an infection, an organ disease, a blood disease, an immune systemdisease, a cancer, a brain and nervous system disease, an endocrinedisease, a pregnancy or childbirth-related disease, an inheriteddisease, or an environmentally-acquired disease, cancer, or a fungalinfection, a bacterial infection, a parasite infection, or a viralinfection.

In certain example embodiments, the RNA-based masking constructsuppresses generation of a detectable positive signal, or the RNA-basedmasking construct suppresses generation of a detectable positive signalby masking the detectable positive signal, or generating a detectablenegative signal instead, or the RNA-based masking construct comprises asilencing RNA that suppresses generation of a gene product encoded by areporting construct, wherein the gene product generates the detectablepositive signal when expressed, or the RNA-based masking construct is aribozyme that generates the negative detectable signal, and wherein thepositive detectable signal is generated when the ribozyme isinactivated. In other example embodiments, the ribozyme converts asubstrate to a first state and wherein the substrate converts to asecond state when the ribozyme is inactivated, or the RNA-based maskingagent is an aptamer, or the aptamer sequesters an enzyme, wherein theenzyme generates a detectable signal upon release from the aptamer byacting upon a substrate, or the aptamer sequesters a pair of agents thatwhen released from the aptamers combine to generate a detectable signal.In still further embodiments, the RNA-based masking construct comprisesan RNA oligonucleotide with a detectable ligand on a first end of theRNA oligonucleotide and a masking component on a second end of the RNAoligonucleotide, or the detectable ligand is a fluorophore and themasking component is a quencher molecule.

With respect to general information on CRISPR-Cas Systems, componentsthereof, and delivery of such components, including methods, materials,delivery vehicles, vectors, particles, AAV, and making and usingthereof, including as to amounts and formulations, all useful in thepractice of the instant invention, reference is made to: U.S. Pat. Nos.8,999,641, 8,993,233, 8,945,839, 8,932,814, 8,906,616, 8,895,308,8,889,418, 8,889,356, 8,871,445, 8,865,406, 8,795,965, 8,771,945 and8,697,359; US Patent Publications US 2014-0310830 (U.S. application Ser.No. 14/105,031), US 2014-0287938 A1 (U.S. application Ser. No.14/213,991), US 2014-0273234 A1 (U.S. App. Ser. No. 14/293,674), US2014-0273232 A1 (U.S. application Ser. No. 14/290,575), US 2014 0273231(U.S. application Ser. No. 14/259,420), US 2014-0256046 A1 (U.S.application Ser. No. 14/226,274), US 2014-0248702 A1 (U.S. applicationSer. No. 14/258,458), US 2014-0242700 A1 (U.S. App. Ser. No.14/222,930), US 2014-0242699 A1 (U.S. application Ser. No. 14/183,512),US 2014-0242664 A1 (U.S. application Ser. No. 14/104,990), US2014-0234972 A1 (U.S. application Ser. No. 14/183,471), US 2014 0227787A1 (U.S. application Ser. No. 14/256,912), US 2014-0189896 A1 (U.S. App.Ser. No. 14/105,035), US 2014-0186958 (U.S. application Ser. No.14/105,017), US 2014-0186919 A1 (U.S. App. Ser. No. 14/104,977), US2014-0186843 A1 (U.S. application Ser. No. 14/104,900), US 2014 0179770A1 (U.S. application Ser. No. 14/104,837) and US 2014-0179006 A1 (U.S.App. Ser. No. 14/183,486), US 2014-0170753 (U.S. application Ser. No.14/183,429); European Patents EP 2 784 162 B1 and EP 2 771 468 B1;European Patent Applications EP 2 771 468 (EP 13 818570.7), EP 2 764 103(EP13824232.6), and EP 2 784 162 (EP14170383.5); and PCT PatentPublications PCT Patent Publications WO 2014/093661 (PCT/US2013/074743),WO 2014/093694 (PCT/US2013/074790), WO 2014/093595 (PCT/US2013/074611),WO 2014/093718 (PCT/US2013/074825), WO 2014/093709 (PCT/US2013/074812),WO 2014/093622 (PCT/US2013/074667), WO 2014/093635 (PCT/US2013/074691),WO 2014/093655 (PCT/US2013/074736), WO 2014/093712 (PCT/US2013/074819),WO 2014/093701 (PCT/US2013/074800), WO 2014/018423 (PCT/US2013/051418),WO 2014/204723 (PCT/US2014/041790), WO 2014/204724 (PCT/US2014/041800),WO 2014/204725 (PCT/US2014/041803), WO 2014/204726 (PCT/US2014/041804),WO 2014/204727 (PCT/US2014/041806), WO 2014/204728 (PCT/US2014/041808),WO 2014/204729 (PCT/US2014/041809). Reference is also made to U.S.provisional patent applications 61/758,468; 61/802,174; 61/806,375;61/814,263; 61/819,803 and 61/828,130, filed on Jan. 30, 2013; Mar. 15,2013; Mar. 28, 2013; Apr. 20, 2013; May 6, 2013 and May 28, 2013respectively. Reference is also made to U.S. provisional patentapplication 61/836,123, filed on Jun. 17, 2013. Reference isadditionally made to U.S. provisional patent applications 61/835,931,61/835,936, 61/836,127, 61/836,101, 61/836,080 and 61/835,973, eachfiled Jun. 17, 2013. Further reference is made to U.S. provisionalpatent applications 61/862,468 and 61/862,355 filed on Aug. 5, 2013;61/871,301 filed on Aug. 28, 2013; 61/960,777 filed on Sep. 25, 2013 and61/961,980 filed on Oct. 28, 2013. Reference is yet further made to: PCTPatent applications Nos: PCT/US2014/041803, PCT/US2014/041800,PCT/US2014/041809, PCT/US2014/041804 and PCT/US2014/041806, each filedJun. 10, 2014 6/10/14; PCT/US2014/041808 filed Jun. 11, 2014; andPCT/US2014/62558 filed Oct. 28, 2014, and U.S. Provisional PatentApplications Ser. Nos. 61/915,150, 61/915,301, 61/915,267 and61/915,260, each filed Dec. 12, 2013; 61/757,972 and 61/768,959, filedon Jan. 29, 2013 and Feb. 25, 2013; 61/835,936, 61/836,127, 61/836,101,61/836,080, 61/835,973, and 61/835,931, filed Jun. 17, 2013; 62/010,888and 62/010,879, both filed Jun. 11, 2014; 62/010,329 and 62/010,441,each filed Jun. 10, 2014; 61/939,228 and 61/939,242, each filed Feb. 12,2014; 61/980,012, filed Apr. 15, 2014; 62/038,358, filed Aug. 17, 2014;62/054,490, 62/055,484, 62/055,460 and 62/055,487, each filed Sep. 25,2014; and 62/069,243, filed Oct. 27, 2014. Reference is also made toU.S. provisional patent applications Nos. 62/055,484, 62/055,460, and62/055,487, filed Sep. 25, 2014; U.S. provisional patent application61/980,012, filed Apr. 15, 2014; and U.S. provisional patent application61/939,242 filed Feb. 12, 2014. Reference is made to PCT applicationdesignating, inter alia, the United States, application No.PCT/US14/41806, filed Jun. 10, 2014. Reference is made to U.S.provisional patent application 61/930,214 filed on Jan. 22, 2014.Reference is made to U.S. provisional patent applications 61/915,251;61/915,260 and 61/915,267, each filed on Dec. 12, 2013. Reference ismade to US provisional patent application U.S. Ser. No. 61/980,012 filedApr. 15, 2014. Reference is made to PCT application designating, interalia, the United States, application No. PCT/US 14/41806, filed Jun. 10,2014. Reference is made to U.S. provisional patent application61/930,214 filed on Jan. 22, 2014. Reference is made to U.S. provisionalpatent applications 61/915,251; 61/915,260 and 61/915,267, each filed onDec. 12, 2013.

Mention is also made of U.S. application 62/091,455, filed, 12 Dec.2014, PROTECTED GUIDE RNAS (PGRNAS); U.S. application 62/096,708, 24Dec. 2014, PROTECTED GUIDE RNAS (PGRNAS); U.S. application 62/091,462,12 Dec. 2014, DEAD GUIDES FOR CRISPR TRANSCRIPTION FACTORS; U.S.application 62/096,324, 23 Dec. 2014, DEAD GUIDES FOR CRISPRTRANSCRIPTION FACTORS; U.S. application 62/091,456, 12 Dec. 2014,ESCORTED AND FUNCTIONALIZED GUIDES FOR CRISPR-CAS SYSTEMS; U.S.application 62/091,461, 12 Dec. 2014, DELIVERY, USE AND THERAPEUTICAPPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR GENOMEEDITING AS TO HEMATOPOIETIC STEM CELLS (HSCs); U.S. application62/094,903, 19 Dec. 14, UNBIASED IDENTIFICATION OF DOUBLE-STRAND BREAKSAND GENOMIC REARRANGEMENT BY GENOME-WISE INSERT CAPTURE SEQUENCING; U.S.application 62/096,761, 24 Dec. 2014, ENGINEERING OF SYSTEMS, METHODSAND OPTIMIZED ENZYME AND GUIDE SCAFFOLDS FOR SEQUENCE MANIPULATION; U.S.application 62/098,059, 30 Dec. 2014, RNA-TARGETING SYSTEM; U.S.application 62/096,656, 24 Dec. 2014, CRISPR HAVING OR ASSOCIATED WITHDESTABILIZATION DOMAINS; U.S. application 62/096,697, 24 Dec. 2014,CRISPR HAVING OR ASSOCIATED WITH AAV; U.S. application 62/098,158, 30Dec. 2014, ENGINEERED CRISPR COMPLEX INSERTIONAL TARGETING SYSTEMS; U.S.application 62/151,052, 22 Apr. 2015, CELLULAR TARGETING FOREXTRACELLULAR EXOSOMAL REPORTING; U.S. application 62/054,490, 24 Sep.2014, DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CASSYSTEMS AND COMPOSITIONS FOR TARGETING DISORDERS AND DISEASES USINGPARTICLE DELIVERY COMPONENTS; U.S. application 62/055,484, 25 Sep. 2014,SYSTEMS, METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION WITHOPTIMIZED FUNCTIONAL CRISPR-CAS SYSTEMS; U.S. application 62/087,537, 4Dec. 2014, SYSTEMS, METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATIONWITH OPTIMIZED FUNCTIONAL CRISPR-CAS SYSTEMS; U.S. application62/054,651, 24 Sep. 2014, DELIVERY, USE AND THERAPEUTIC APPLICATIONS OFTHE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR MODELING COMPETITION OFMULTIPLE CANCER MUTATIONS IN VIVO; U.S. application 62/067,886, 23 Oct.2014, DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CASSYSTEMS AND COMPOSITIONS FOR MODELING COMPETITION OF MULTIPLE CANCERMUTATIONS IN VIVO; U.S. application 62/054,675, 24 Sep. 2014, DELIVERY,USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CAS SYSTEMS ANDCOMPOSITIONS IN NEURONAL CELLS/TISSUES; U.S. application 62/054,528, 24Sep. 2014, DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CASSYSTEMS AND COMPOSITIONS IN IMMUNE DISEASES OR DISORDERS; U.S.application 62/055,454, 25 Sep. 2014, DELIVERY, USE AND THERAPEUTICAPPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR TARGETINGDISORDERS AND DISEASES USING CELL PENETRATION PEPTIDES (CPP); U.S.application 62/055,460, 25 Sep. 2014, MULTIFUNCTIONAL-CRISPR COMPLEXESAND/OR OPTIMIZED ENZYME LINKED FUNCTIONAL-CRISPR COMPLEXES; U.S.application 62/087,475, 4 Dec. 2014, FUNCTIONAL SCREENING WITH OPTIMIZEDFUNCTIONAL CRISPR-CAS SYSTEMS; U.S. application 62/055,487, 25 Sep.2014, FUNCTIONAL SCREENING WITH OPTIMIZED FUNCTIONAL CRISPR-CAS SYSTEMS;U.S. application 62/087,546, 4 Dec. 2014, MULTIFUNCTIONAL CRISPRCOMPLEXES AND/OR OPTIMIZED ENZYME LINKED FUNCTIONAL-CRISPR COMPLEXES;and U.S. application 62/098,285, 30 Dec. 2014, CRISPR MEDIATED IN VIVOMODELING AND GENETIC SCREENING OF TUMOR GROWTH AND METASTASIS.

Each of these patents, patent publications, and applications, and alldocuments cited therein or during their prosecution (“appln citeddocuments”) and all documents cited or referenced in the appln citeddocuments, together with any instructions, descriptions, productspecifications, and product sheets for any products mentioned therein orin any document therein and incorporated by reference herein, are herebyincorporated herein by reference, and may be employed in the practice ofthe invention. All documents (e.g., these patents, patent publicationsand applications and the appln cited documents) are incorporated hereinby reference to the same extent as if each individual document wasspecifically and individually indicated to be incorporated by reference.

Also with respect to general information on CRISPR-Cas Systems, mentionis made of the following (also hereby incorporated herein by reference):

-   Multiplex genome engineering using CRISPR/Cas systems. Cong, L.,    Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P. D.,    Wu, X., Jiang, W., Marraffini, L. A., & Zhang, F. Science February    15; 339(6121):819-23 (2013);-   RNA-guided editing of bacterial genomes using CRISPR-Cas systems.    Jiang W., Bikard D., Cox D., Zhang F, Marraffini L A. Nat Biotechnol    March; 31(3):233-9 (2013);-   One-Step Generation of Mice Carrying Mutations in Multiple Genes by    CRISPR/Cas-Mediated Genome Engineering. Wang H., Yang H., Shivalila    C S., Dawlaty M M., Cheng A W., Zhang F., Jaenisch R. Cell May 9;    153(4):910-8 (2013);-   Optical control of mammalian endogenous transcription and epigenetic    states. Konermann S, Brigham M D, Trevino A E, Hsu P D, Heidenreich    M, Cong L, Platt R J, Scott D A, Church G M, Zhang F. Nature. August    22; 500(7463):472-6. doi: 10.1038/Nature12466. Epub 2013 Aug. 23    (2013);-   Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome Editing    Specificity. Ran, F A., Hsu, P D., Lin, C Y., Gootenberg, J S.,    Konermann, S., Trevino, A E., Scott, D A., Inoue, A., Matoba, S.,    Zhang, Y., & Zhang, F. Cell August 28. pii: 50092-8674(13)01015-5    (2013-A);-   DNA targeting specificity of RNA-guided Cas9 nucleases. Hsu, P.,    Scott, D., Weinstein, J., Ran, F A., Konermann, S., Agarwala, V.,    Li, Y., Fine, E., Wu, X., Shalem, O., Cradick, T J., Marraffini, L    A., Bao, G., & Zhang, F. Nat Biotechnol doi:10.1038/nbt.2647 (2013);-   Genome engineering using the CRISPR-Cas9 system. Ran, F A., Hsu, P    D., Wright, J., Agarwala, V., Scott, DA., Zhang, F. Nature Protocols    November; 8(11):2281-308 (2013-B);-   Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells. Shalem,    O., Sanjana, N E., Hartenian, E., Shi, X., Scott, D A., Mikkelson,    T., Heckl, D., Ebert, B L., Root, D E., Doench, J G., Zhang, F.    Science Dec. 12. (2013). [Epub ahead of print];-   Crystal structure of cas9 in complex with guide RNA and target DNA.    Nishimasu, H., Ran, F A., Hsu, P D., Konermann, S., Shehata, S I.,    Dohmae, N., Ishitani, R., Zhang, F., Nureki, O. Cell February 27,    156(5):935-49 (2014);-   Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian    cells. Wu X., Scott D A., Kriz A J., Chiu A C., Hsu P D., Dadon D    B., Cheng A W., Trevino A E., Konermann S., Chen S., Jaenisch R.,    Zhang F., Sharp P A. Nat Biotechnol. April 20. doi: 10.1038/nbt.2889    (2014);-   CRISPR-Cas9 Knockin Mice for Genome Editing and Cancer Modeling.    Platt R J, Chen S, Zhou Y, Yim M J, Swiech L, Kempton H R, Dahlman J    E, Parnas O, Eisenhaure T M, Jovanovic M, Graham D B, Jhunjhunwala    S, Heidenreich M, Xavier R J, Langer R, Anderson D G, Hacohen N,    Regev A, Feng G, Sharp P A, Zhang F. Cell 159(2): 440-455 DOI:    10.1016/j.cell.2014.09.014(2014);-   Development and Applications of CRISPR-Cas9 for Genome Engineering,    Hsu P D, Lander E S, Zhang F., Cell. June 5; 157(6):1262-78 (2014).-   Genetic screens in human cells using the CRISPR/Cas9 system, Wang T,    Wei J J, Sabatini D M, Lander E S., Science. January 3; 343(6166):    80-84. doi:10.1126/science.1246981 (2014);-   Rational design of highly active sgRNAs for CRISPR-Cas9-mediated    gene inactivation, Doench J G, Hartenian E, Graham D B, Tothova Z,    Hegde M, Smith I, Sullender M, Ebert B L, Xavier R J, Root D E.,    (published online 3 Sep. 2014) Nat Biotechnol. December;    32(12):1262-7 (2014);-   In vivo interrogation of gene function in the mammalian brain using    CRISPR-Cas9, Swiech L, Heidenreich M, Banerjee A, Habib N, Li Y,    Trombetta J, Sur M, Zhang F., (published online 19 Oct. 2014) Nat    Biotechnol. January; 33(1):102-6 (2015);-   Genome-scale transcriptional activation by an engineered CRISPR-Cas9    complex, Konermann S, Brigham M D, Trevino A E, Joung J, Abudayyeh O    O, Barcena C, Hsu P D, Habib N, Gootenberg J S, Nishimasu H, Nureki    O, Zhang F., Nature. January 29; 517(7536):583-8 (2015).-   A split-Cas9 architecture for inducible genome editing and    transcription modulation, Zetsche B, Volz S E, Zhang F., (published    online 2 Feb. 2015) Nat Biotechnol. February; 33(2):139-42 (2015);-   Genome-wide CRISPR Screen in a Mouse Model of Tumor Growth and    Metastasis, Chen S, Sanjana N E, Zheng K, Shalem O, Lee K, Shi X,    Scott D A, Song J, Pan J Q, Weissleder R, Lee H, Zhang F, Sharp P A.    Cell 160, 1246-1260, Mar. 12, 2015 (multiplex screen in mouse), and-   In vivo genome editing using Staphylococcus aureus Cas9, Ran F A,    Cong L, Yan W X, Scott D A, Gootenberg J S, Kriz A J, Zetsche B,    Shalem O, Wu X, Makarova K S, Koonin E V, Sharp P A, Zhang F.,    (published online 1 Apr. 2015), Nature. April 9; 520(7546):186 91    (2015).-   Shalem et al., “High-throughput functional genomics using    CRISPR-Cas9,” Nature Reviews Genetics 16, 299-311 (May 2015).-   Xu et al., “Sequence determinants of improved CRISPR sgRNA design,”    Genome Research 25, 1147-1157 (August 2015).-   Parnas et al., “A Genome-wide CRISPR Screen in Primary Immune Cells    to Dissect Regulatory Networks,” Cell 162, 675-686 (Jul. 30, 2015).-   Ramanan et al., CRISPR/Cas9 cleavage of viral DNA efficiently    suppresses hepatitis B virus,” Scientific Reports 5:10833. doi:    10.1038/srep10833 (Jun. 2, 2015).-   Nishimasu et al., Crystal Structure of Staphylococcus aureus Cas9,”    Cell 162, 1113-1126 (Aug. 27, 2015).-   Zetsche et al. (2015), “Cpf1 is a single RNA-guided endonuclease of    a class 2 CRISPR-Cas system,” Cell 163, 759-771 (Oct. 22, 2015) doi:    10.1016/j.cell.2015.09.038. Epub Sep. 25, 2015.-   Shmakov et al. (2015), “Discovery and Functional Characterization of    Diverse Class 2 CRISPR-Cas Systems,” Molecular Cell 60, 385-397    (Nov. 5, 2015) doi: 10.1016/j.molcel.2015.10.008. Epub Oct. 22,    2015.-   Dahlman et al., “Orthogonal gene control with a catalytically active    Cas9 nuclease,” Nature Biotechnology 33, 1159-1161 (November, 2015).-   Gao et al, “Engineered Cpf1 Enzymes with Altered PAM Specificities,”    bioRxiv 091611; doi: http://dx.doi.org/10.1101/091611 Epub Dec. 4,    2016.-   Smargon et al. (2017), “Cas13b Is a Type VI-B CRISPR-Associated    RNA-Guided RNase Differentially Regulated by Accessory Proteins    Csx27 and Csx28,” Molecular Cell 65, 618-630 (Feb. 16, 2017) doi:    10.1016/j.molcel.2016.12.023. Epub Jan. 5, 2017.    each of which is incorporated herein by reference, may be considered    in the practice of the instant invention, and discussed briefly    below:-   Cong et al. engineered type II CRISPR-Cas systems for use in    eukaryotic cells based on both Streptococcus thermophilus Cas9 and    also Streptococcus pyogenes Cas9 and demonstrated that Cas9    nucleases can be directed by short RNAs to induce precise cleavage    of DNA in human and mouse cells. Their study further showed that    Cas9 as converted into a nicking enzyme can be used to facilitate    homology-directed repair in eukaryotic cells with minimal mutagenic    activity. Additionally, their study demonstrated that multiple guide    sequences can be encoded into a single CRISPR array to enable    simultaneous editing of several at endogenous genomic loci sites    within the mammalian genome, demonstrating easy programmability and    wide applicability of the RNA-guided nuclease technology. This    ability to use RNA to program sequence specific DNA cleavage in    cells defined a new class of genome engineering tools. These studies    further showed that other CRISPR loci are likely to be    transplantable into mammalian cells and can also mediate mammalian    genome cleavage. Importantly, it can be envisaged that several    aspects of the CRISPR-Cas system can be further improved to increase    its efficiency and versatility.-   Jiang et al. used the clustered, regularly interspaced, short    palindromic repeats (CRISPR)-associated Cas9 endonuclease complexed    with dual-RNAs to introduce precise mutations in the genomes of    Streptococcus pneumoniae and Escherichia coli. The approach relied    on dual-RNA:Cas9-directed cleavage at the targeted genomic site to    kill unmutated cells and circumvents the need for selectable markers    or counter-selection systems. The study reported reprogramming    dual-RNA:Cas9 specificity by changing the sequence of short CRISPR    RNA (crRNA) to make single- and multinucleotide changes carried on    editing templates. The study showed that simultaneous use of two    crRNAs enabled multiplex mutagenesis. Furthermore, when the approach    was used in combination with recombineering, in S. pneumoniae,    nearly 100% of cells that were recovered using the described    approach contained the desired mutation, and in E. coli, 65% that    were recovered contained the mutation.-   Wang et al. (2013) used the CRISPR/Cas system for the one-step    generation of mice carrying mutations in multiple genes which were    traditionally generated in multiple steps by sequential    recombination in embryonic stem cells and/or time-consuming    intercrossing of mice with a single mutation. The CRISPR/Cas system    will greatly accelerate the in vivo study of functionally redundant    genes and of epistatic gene interactions.-   Konermann et al. (2013) addressed the need in the art for versatile    and robust technologies that enable optical and chemical modulation    of DNA-binding domains based CRISPR Cas9 enzyme and also    Transcriptional Activator Like Effectors.-   Ran et al. (2013-A) described an approach that combined a Cas9    nickase mutant with paired guide RNAs to introduce targeted    double-strand breaks. This addresses the issue of the Cas9 nuclease    from the microbial CRISPR-Cas system being targeted to specific    genomic loci by a guide sequence, which can tolerate certain    mismatches to the DNA target and thereby promote undesired    off-target mutagenesis. Because individual nicks in the genome are    repaired with high fidelity, simultaneous nicking via appropriately    offset guide RNAs is required for double-stranded breaks and extends    the number of specifically recognized bases for target cleavage. The    authors demonstrated that using paired nicking can reduce off-target    activity by 50- to 1,500-fold in cell lines and to facilitate gene    knockout in mouse zygotes without sacrificing on-target cleavage    efficiency. This versatile strategy enables a wide variety of genome    editing applications that require high specificity.-   Hsu et al. (2013) characterized SpCas9 targeting specificity in    human cells to inform the selection of target sites and avoid    off-target effects. The study evaluated>700 guide RNA variants and    SpCas9-induced indel mutation levels at >100 predicted genomic    off-target loci in 293T and 293FT cells. The authors that SpCas9    tolerates mismatches between guide RNA and target DNA at different    positions in a sequence-dependent manner, sensitive to the number,    position and distribution of mismatches. The authors further showed    that SpCas9-mediated cleavage is unaffected by DNA methylation and    that the dosage of SpCas9 and sgRNA can be titrated to minimize    off-target modification. Additionally, to facilitate mammalian    genome engineering applications, the authors reported providing a    web-based software tool to guide the selection and validation of    target sequences as well as off-target analyses.-   Ran et al. (2013-B) described a set of tools for Cas9-mediated    genome editing via non-homologous end joining (NHEJ) or    homology-directed repair (HDR) in mammalian cells, as well as    generation of modified cell lines for downstream functional studies.    To minimize off-target cleavage, the authors further described a    double-nicking strategy using the Cas9 nickase mutant with paired    guide RNAs. The protocol provided by the authors experimentally    derived guidelines for the selection of target sites, evaluation of    cleavage efficiency and analysis of off-target activity. The studies    showed that beginning with target design, gene modifications can be    achieved within as little as 1-2 weeks, and modified clonal cell    lines can be derived within 2-3 weeks.-   Shalem et al. described a new way to interrogate gene function on a    genome-wide scale. Their studies showed that delivery of a    genome-scale CRISPR-Cas9 knockout (GeCKO) library targeted 18,080    genes with 64,751 unique guide sequences enabled both negative and    positive selection screening in human cells. First, the authors    showed use of the GeCKO library to identify genes essential for cell    viability in cancer and pluripotent stem cells. Next, in a melanoma    model, the authors screened for genes whose loss is involved in    resistance to vemurafenib, a therapeutic that inhibits mutant    protein kinase BRAF. Their studies showed that the highest-ranking    candidates included previously validated genes NF1 and MED12 as well    as novel hits NF2, CUL3, TADA2B, and TADA1. The authors observed a    high level of consistency between independent guide RNAs targeting    the same gene and a high rate of hit confirmation, and thus    demonstrated the promise of genome-scale screening with Cas9.-   Nishimasu et al. reported the crystal structure of Streptococcus    pyogenes Cas9 in complex with sgRNA and its target DNA at 2.5 A°    resolution. The structure revealed a bibbed architecture composed of    target recognition and nuclease lobes, accommodating the sgRNA:DNA    heteroduplex in a positively charged groove at their interface.    Whereas the recognition lobe is essential for binding sgRNA and DNA,    the nuclease lobe contains the HNH and RuvC nuclease domains, which    are properly positioned for cleavage of the complementary and    non-complementary strands of the target DNA, respectively. The    nuclease lobe also contains a carboxyl-terminal domain responsible    for the interaction with the protospacer adjacent motif (PAM). This    high-resolution structure and accompanying functional analyses have    revealed the molecular mechanism of RNA-guided DNA targeting by    Cas9, thus paving the way for the rational design of new, versatile    genome-editing technologies.-   Wu et al. mapped genome-wide binding sites of a catalytically    inactive Cas9 (dCas9) from Streptococcus pyogenes loaded with single    guide RNAs (sgRNAs) in mouse embryonic stem cells (mESCs). The    authors showed that each of the four sgRNAs tested targets dCas9 to    between tens and thousands of genomic sites, frequently    characterized by a 5-nucleotide seed region in the sgRNA and an NGG    protospacer adjacent motif (PAM). Chromatin inaccessibility    decreases dCas9 binding to other sites with matching seed sequences;    thus 70% of off-target sites are associated with genes. The authors    showed that targeted sequencing of 295 dCas9 binding sites in mESCs    transfected with catalytically active Cas9 identified only one site    mutated above background levels. The authors proposed a two-state    model for Cas9 binding and cleavage, in which a seed match triggers    binding but extensive pairing with target DNA is required for    cleavage.-   Platt et al. established a Cre-dependent Cas9 knockin mouse. The    authors demonstrated in vivo as well as ex vivo genome editing using    adeno-associated virus (AAV)-, lentivirus-, or particle-mediated    delivery of guide RNA in neurons, immune cells, and endothelial    cells.-   Hsu et al. (2014) is a review article that discusses generally    CRISPR-Cas9 history from yogurt to genome editing, including genetic    screening of cells.-   Wang et al. (2014) relates to a pooled, loss-of-function genetic    screening approach suitable for both positive and negative selection    that uses a genome-scale lentiviral single guide RNA (sgRNA)    library.-   Doench et al. created a pool of sgRNAs, tiling across all possible    target sites of a panel of six endogenous mouse and three endogenous    human genes and quantitatively assessed their ability to produce    null alleles of their target gene by antibody staining and flow    cytometry. The authors showed that optimization of the PAM improved    activity and also provided an on-line tool for designing sgRNAs.-   Swiech et al. demonstrate that AAV-mediated SpCas9 genome editing    can enable reverse genetic studies of gene function in the brain.-   Konermann et al. (2015) discusses the ability to attach multiple    effector domains, e.g., transcriptional activator, functional and    epigenomic regulators at appropriate positions on the guide such as    stem or tetraloop with and without linkers.-   Zetsche et al. demonstrates that the Cas9 enzyme can be split into    two and hence the assembly of Cas9 for activation can be controlled.-   Chen et al. relates to multiplex screening by demonstrating that a    genome-wide in vivo CRISPR-Cas9 screen in mice reveals genes    regulating lung metastasis.-   Ran et al. (2015) relates to SaCas9 and its ability to edit genomes    and demonstrates that one cannot extrapolate from biochemical    assays. Shalem et al. (2015) described ways in which catalytically    inactive Cas9 (dCas9) fusions are used to synthetically repress    (CRISPRi) or activate (CRISPRa) expression, showing. advances using    Cas9 for genome-scale screens, including arrayed and pooled screens,    knockout approaches that inactivate genomic loci and strategies that    modulate transcriptional activity.

End Edits

-   Shalem et al. (2015) described ways in which catalytically inactive    Cas9 (dCas9) fusions are used to synthetically repress (CRISPRi) or    activate (CRISPRa) expression, showing. advances using Cas9 for    genome-scale screens, including arrayed and pooled screens, knockout    approaches that inactivate genomic loci and strategies that modulate    transcriptional activity.-   Xu et al. (2015) assessed the DNA sequence features that contribute    to single guide RNA (sgRNA) efficiency in CRISPR-based screens. The    authors explored efficiency of CRISPR/Cas9 knockout and nucleotide    preference at the cleavage site. The authors also found that the    sequence preference for CRISPRi/a is substantially different from    that for CRISPR/Cas9 knockout.-   Parnas et al. (2015) introduced genome-wide pooled CRISPR-Cas9    libraries into dendritic cells (DCs) to identify genes that control    the induction of tumor necrosis factor (Tnf) by bacterial    lipopolysaccharide (LPS). Known regulators of Tlr4 signaling and    previously unknown candidates were identified and classified into    three functional modules with distinct effects on the canonical    responses to LPS.-   Ramanan et al (2015) demonstrated cleavage of viral episomal DNA    (cccDNA) in infected cells. The HBV genome exists in the nuclei of    infected hepatocytes as a 3.2 kb double-stranded episomal DNA    species called covalently closed circular DNA (cccDNA), which is a    key component in the HBV life cycle whose replication is not    inhibited by current therapies. The authors showed that sgRNAs    specifically targeting highly conserved regions of HBV robustly    suppresses viral replication and depleted cccDNA.-   Nishimasu et al. (2015) reported the crystal structures of SaCas9 in    complex with a single guide RNA (sgRNA) and its double-stranded DNA    targets, containing the 5′-TTGAAT-3′ PAM and the 5′-TTGGGT-3′ PAM. A    structural comparison of SaCas9 with SpCas9 highlighted both    structural conservation and divergence, explaining their distinct    PAM specificities and orthologous sgRNA recognition.

Also, “Dimeric CRISPR RNA-guided FokI nucleases for highly specificgenome editing”, Shengdar Q. Tsai, Nicolas Wyvekens, Cyd Khayter,Jennifer A. Foden, Vishal Thapar, Deepak Reyon, Mathew J. Goodwin,Martin J. Aryee, J. Keith Joung Nature Biotechnology 32(6): 569-77(2014), relates to dimeric RNA-guided FokI Nucleases that recognizeextended sequences and can edit endogenous genes with high efficienciesin human cells. In addition, mention is made of PCT applicationPCT/US14/70057, Attorney Reference 47627.99.2060 and BI-2013/107entitled “DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CASSYSTEMS AND COMPOSITIONS FOR TARGETING DISORDERS AND DISEASES USINGPARTICLE DELIVERY COMPONENTS (claiming priority from one or more or allof US provisional patent applications: 62/054,490, filed Sep. 24, 2014;62/010,441, filed Jun. 10, 2014; and 61/915,118, 61/915,215 and61/915,148, each filed on Dec. 12, 2013) (“the Particle Delivery PCT”),incorporated herein by reference, with respect to a method of preparingan sgRNA-and-Cas9 protein containing particle comprising admixing amixture comprising an sgRNA and Cas9 protein (and optionally HDRtemplate) with a mixture comprising or consisting essentially of orconsisting of surfactant, phospholipid, biodegradable polymer,lipoprotein and alcohol; and particles from such a process. For example,wherein Cas9 protein and sgRNA were mixed together at a suitable, e.g.,3:1 to 1:3 or 2:1 to 1:2 or 1:1 molar ratio, at a suitable temperature,e.g., 15-30C, e.g., 20-25C, e.g., room temperature, for a suitable time,e.g., 15-45, such as 30 minutes, advantageously in sterile, nucleasefree buffer, e.g., 1×PBS. Separately, particle components such as orcomprising: a surfactant, e.g., cationic lipid, e.g.,1,2-dioleoyl-3-trimethylammonium-propane (DOTAP); phospholipid, e.g.,dimyristoylphosphatidylcholine (DMPC); biodegradable polymer, such as anethylene-glycol polymer or PEG, and a lipoprotein, such as a low-densitylipoprotein, e.g., cholesterol were dissolved in an alcohol,advantageously a C₁₋₆ alkyl alcohol, such as methanol, ethanol,isopropanol, e.g., 100% ethanol. The two solutions were mixed togetherto form particles containing the Cas9-sgRNA complexes. Accordingly,sgRNA may be pre-complexed with the Cas9 protein, before formulating theentire complex in a particle. Formulations may be made with a differentmolar ratio of different components known to promote delivery of nucleicacids into cells (e.g. 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP),1,2-ditetradecanoyl-sn-glycero-3-phosphocholine (DMPC), polyethyleneglycol (PEG), and cholesterol) For example DOTAP: DMPC: PEG: CholesterolMolar Ratios may be DOTAP 100, DMPC 0, PEG 0, Cholesterol 0; or DOTAP90, DMPC 0, PEG 10, Cholesterol 0; or DOTAP 90, DMPC 0, PEG 5,Cholesterol 5. DOTAP 100, DMPC 0, PEG 0, Cholesterol 0. That applicationaccordingly comprehends admixing sgRNA, Cas9 protein and components thatform a particle; as well as particles from such admixing. Aspects of theinstant invention can involve particles; for example, particles using aprocess analogous to that of the Particle Delivery PCT, e.g., byadmixing a mixture comprising crRNA and/or Cas13b as in the instantinvention and components that form a particle, e.g., as in the ParticleDelivery PCT, to form a particle and particles from such admixing (or,of course, other particles involving crRNA and/or Cas13b as in theinstant invention).

The present invention will be further illustrated in the followingExamples which are given for illustration purposes only and are notintended to limit the invention in any way.

EXAMPLES Example 1: Identification of Cas13b Orthologs

Cas13b proteins shown in Table 1A and Table 1B below are advantageouslyfrom codon optimization for expression in mammalian cells. Fusionconstructs of each of the Cas13b orthologues with mCherry and optionallyNLS or NES are made and cloned in a mammalian expression vector. Thevarious Cas13b orthologues are transfected in HEK293T cells and cellularlocalization is evaluated based on mCherry expression. Localizations ofdifferent Cas13b orthologues fused to a C-terminal and N-terminal NES,fused to a C-terminal and N-terminal NLS, or without NES or NLS fusionare determined. NES fusions efficiently result in cytoplasmiclocalization of the Cas13b protein. NLS fusions efficiently result innuclear localization of the Cas13b protein. Variably, also nucleolarlocalization can be observed with NLS fusions.

TABLE 1A Sinomicrobium WP_072319476.1MESTTTLGLHLKYQHDLFEDKHYFGGGVNLAVQNIESIFQAFA oceaniERYGIQNPLRKNGVPAINNIFHDNISISNYKEYLKFLKQYLPVVGFLEKSNEINIFEFREDFEILINAIYKLRHFYTHYYHSPIKLEDRFYTCLNELFVAVAIQVKKHKMKSDKTRQLLNKNLHQLLQQLIEQKREKLKDKKAEGEKVSLDTKSIENAVLNDAFVHLLDKDENIRLNYSSRLSEDIITKNGITLSISGLLFLLSLFLQRKEAEDLRSRIEGFKGKGNELRFMATHWVFSYLNVKRIKHRLNTDFQKETLLIQIADELSKVPDEVYKTLDHENRSKFLEDINEYIREGNEDASLNESTVVHGVIRKRYENKFHYLVLRYLDEFVDFPSLRFQVHLGNYIHDRRDKVIDGTNFITNRVIKEPIKVFGKLSHVSKLKSDYMESLSREHKNGWDVFPNPSYNFVGHNIPIFINLRSASSKGKELYRDLMKIKSEKKKKSREEGIPMERRDGKPTKIEISNQIDRNIKDNNFKDIYPGEPLAMLSLNELPALLFELLRRPSITPQDIEDRMVEKLYERFQIIRDYKPGDGLSTSKISKKLRKADNSTRLDGKKLLRAIQTETRNAREKLHTLEENKALQKNRKRRTVYTTREQGREASWLAQDLKRFMPIASRKEWRGYHHSQLQQILAFYDQNPKQPLELLEQFWDLKEDTYVWNSWIHKSLSQHNGFVPMYEGYLKGRLGYYKKLESDIIGFLEEHKVLKRYYTQQHLNVIFRERLYFIKTETKQKLELLARPLVFPRGIFDDKPTFVQDKKVVDHPELFADWYVYSYKDDHSFQEFYHYKRDYNEIFETELSWDIDFKDNKRQLNPSEQMDLFRMKWDLKIKKIKIQDIFLKIVAEDIYLKIFGHKIPLSLSDFYISRQERLTLDEQAVAQSMRLPGDTSENQIKESNLWQTTVPYEKEQIREPKIKLKDIGKFKYFLQQQKVLNLLKYDPQHVWTKAELEEELYIGKHSYEVVRREMLLQKCHQLEKHILEQFRFDGSNHPRELEQGNHPNFKMYIVNGILTKRGELEIEAENWWLELGNSKNSLDKVEVELLTMKTIPEQKAFLLILIRNKFAHNQLPADNYFHYASNLMNLKKSDTYSLFWFTVADTIVQ EFMSL (SEQ ID NO: 31)Prevotella 12 MEDDKKTTDSIRYELKDKHFWAAFLNLARHNVYITVNHINKIL intermediaEEDEINRDGYENTLENSWNEIKDINKKDRLSKLIIKHFPFLEATTYRQNPTDTTKQKEEKQAEAQSLESLKKSFFVFIYKLRDLRNHYSHYKHSKSLERPKFEEDLQNKMYNIFDVSIQFVKEDYKHNTDINPKKDFKHLDRKRKGKFHYSFADNEGNITESGLLFFVSLFLEKKDAIWVQKKLEGFKCSNKSYQKMTNEVFCRSRMLLPKLRLESTQTQDWILLDMLNELIRCPKSLYERLQGVNRKKFYVSFDPADEDYDAEQEPFKNTLVRHQDRFPYFALRYFDYNEVFANLRFQIDLGTYHFSIYKKLIGGQKEDRHLTHKLYGFERIQEFDKQNRPDEWKAIVKDSDTFKKKEEKEEEKPYISETTPHYHLENKKIGIAFKNHNIWPSTQTELTNNKRKKYNLGTSIKAEAFLSVHELLPMMFYYLLLKTENTKNDNKVGGKKETKKQGKHKIEAIIESKIKDIYALYDAFANGEINSEDELKEYLKGKDIKIVHLPKQMIAILKNEHKDMAEKAEAKQEKMKLATENRLKTLDKQLKGKIQNGKRYNSAPKSGEIASWLVNDMMRFQPVQKDENGESLNNSKANSTEYQLLQRTLAFFGSEHERLAPYFKQTKLIESSNPHPFLNDTEWEKCSNILSFYRSYLKARKNFLESLKPEDWEKNQYFLMLKEPKTNRETLVQGWKNGFNLPRGFFTEPIRKWFMEHWKSIKVDDLKRVGLVAKVTPLFFSEKYKDSVQPFYNYPFNVGDVNKPKEEDFLHREERIELWDKKKDKFKGYKAKKKFKEMTDKEKEEHRSYLEFQSWNKFERELRLVRNQDIVTWLLCTELIDKLKIDELNIKELKKLRLKDINTDTAKKEKNNILNRVMPMELPVTVYKVNKGGYIIKNKPLHTIYIKEAETKLLKQGNFKALVKDRRLNGLFSFVKTPSEAESESNPISKLRVEYELGKYQNARLDIIEDMLALEKKLIDKYNSLDTDNFHNMLTGWLELKGEAKKARFQNDVKLLTAVRNAFSHNQYPMYDENLFGNIERFSLSSSNIIESKGLDIAAKLKEEVSKAAKKIQNEEDNKKEKET (SEQ ID NO: 32) Porphyromonas 19MTEQNEKPYNGTYYTLEDKHFWAAFLNLARHNAYITLAHIDR gingivalisQLAYSKADITNDEDILFFKGQWKNLDNDLERKARLRSLILKHFSFLEGAAYGKKLFESQSSGNKSSKKKELSKKEKEELQANALSLDNLKSILFDFLQKLKDFRNYYSHYRHPESSELPLFDGNMLQRLYNVFDVSVQRVKRDHEHNDKVDPHRHFNHLVRKGKKDKYGNNDNPFFKHHFVDREGTVTEAGLLFFVSLFLEKRDAIWMQKKIRGFKGGTEAYQQMTNEVFCRSRISLPKLKLESLRTDDWMLLDMLNELVRCPKSLYDRLREEDRARFRVPVDILSDEDDTDGTEEDPFKNTLVRHQDRFPYFALRYFDLKKVFTSLRFHIDLGTYHFAIYKKNIGEQPEDRHLTRNLYGFGRIQDFAEEHRPEEWKRLVRDLDYFETGDKPYITQTTPHYHIEKGKIGLRFVPEGQHLWPSPEVGATRTGRSKYAQDKRLTAEAFLSVHELMPMMFYYFLLREKYSEEVSAEKVQGRIKRVIEDVYAVYDAFARDEINTRDELDACLADKGIRRGHLPRQMIAILSQEHKDMEEKVRKKLQEMIADTDHRLDMLDRQTDRKIRIGRKNAGLPKSGVVADWLVRDMMRFQPVAKDTSGKPLNNSKANSTEYRMLQRALALFGGEKERLTPYFRQMNLTGGNNPHPFLHETRWESHTNILSFYRSYLEARKAFLQSIGRSDRVENHRFLLLKEPKTDRQTLVAGWKGEFHLPRGIFTEAVRDCLIEMGYDEVGSYKEVGFMAKAVPLYFERASKDRVQPFYDYPFNVGNSLKPKKGRFLSKEKRAEEWESGKERFRLAKLKKEILEAKEHPYHDFKSWQKFERELRLVKNQDIITWMMCRDLMEENKVEGLDTGTLYLKDIRTDVQEQGSLNVLNRVKPMRLPVVVYRADSRGHVHKEQAPLATVYIEERDTKLLKQGNFKSFVKDRRLNGLFSFVDTGALAMEQYPISKLRVEYELAKYQTARVCAFEQTLELEESLLTRYPHLPDKNFRKMLESWSDPLLDKWPDLHGNVRLLIAVRNAFSHNQYPMYDETLFSSIRKYDPSSPDAIEERMGLNIAHRLSEEVKQAKEMVERIIQA (SEQ ID NO: 33) A2033_10205OFX18020.1 MENQTQKGKGIYYYYTKNEDKHYFGSFLNLANNNIEQIIEEFRI [BacteroidetesRLSLKDEKNIKEIINNYFTDKKSYTDWERGINILKEYLPVIDYLD bacteriumLAITDKEFEKIDLKQKETAKRKYFRTNFSLLIDTIIDLRNFYTHYF GWA2_31_9]HKPISINPDVAKFLDKNLLNVCLDIKKQKMKTDKTKQALKDGLDKELKKLIELKKAELKEKKIKTWNITENVEGAVYNDAFNHMVYKNNAGVTILKDYHKSILPDDKIDSELKLNFSISGLVFLLSMFLSKKEIEQFKSNLEGFKGKVIGENGEYEISKFNNSLKYMATHWIFSYLTFKGLKQRVKNTFDKETLLMQMIDELNKVPHEVYQTLSKEQQNEFLEDINEYVQDNEENKKSMENSIVVHPVIRKRYDDKFNYFAIRFLDEFANFPTLKFFVTAGNFVHDKREKQIQGSMLTSDRMIKEKINVFGKLTEIAKYKSDYFSNENTLETSEWELFPNPSYLLIQNNIPVHIDLIHNTEEAKQCQIAIDRIKCTTNPAKKRNTRKSKEEIIKIIYQKNKNIKYGDPTALLSSNELPALIYELLVNKKSGKELENIIVEKIVNQYKTIAGFEKGQNLSNSLITKKLKKSEPNEDKINAEKIILAINRELEITENKLNIIKNNRAEFRTGAKRKHIFYSKELGQEATWIAYDLKRFMPEASRKEWKGFHHSELQKFLAFYDRNKNDAKALLNMFWNFDNDQLIGNDLNSAFREFHFDKFYEKYLIKRDEILEGFKSFISNFKDEPKLLKKGIKDIYRVFDKRYYIIKSTNAQKEQLLSKPICLPRGIFDNKPTYIEGVKVESNSALFADWYQYTYSDKHEFQSFYDMPRDYKEQFEKFELNNIKSIQNKKNLNKSDKFIYFRYKQDLKIKQIKSQDLFIKLMVDELFNVVFKNNIELNLKKLYQTSDERFKNQLIADVQKNREKGDTSDNKMNENFIWNMTIPLSLCNGQIEEPKVKLKDIGKFRKLETDDKVIQLLEYDKSKVWKKLEIEDELENMPNSYERIRREKLLKGIQEFEHFLLEKEKFDGINHPKHFEQDLNPNFKTYVINGVLRKNSKLNYTEIDKLLDLEHISIKDIETSAKEIHLAYFLIHVRNKFGHNQLPKLEAFELMKKYYKKNNEETYAEYFHKVSSQIVNEF KNSLEKHS (SEQ ID NO: 34SAMN054 SDI27289.1 MEKTQTGLGIYYDHTKLQDKYFFGGFFNLAQNNIDNVIKAFIIK21542_0666 FFPERKDKDINIAQFLDICFKDNDADSDFQKKNKFLRIHFPVIGF [Chryseo-LTSDNDKAGFKKKFALLLKTISELRNFYTHYYHKSIEFPSELFEL bacteriumLDDIFVKTTSEIKKLKKKDDKTQQLLNKNLSEEYDIRYQQQIER jejuense]LKELKAQGKRVSLTDETAIRNGVFNAAFNHLIYRDGENVKPSRLYQSSYSEPDPAENGISLSQNSILFLLSMFLERKETEDLKSRVKGFKAKIIKQGEEQISGLKFMATHWVFSYLCFKGIKQKLSTEFHEETLLIQIIDELSKVPDEVYSAFDSKTKEKFLEDINEYMKEGNADLSLEDSKVIHPVIRKRYENKFNYFAIRFLDEYLSSTSLKFQVHVGNYVHDRRVKHINGTGFQTERIVKDRIKVFGRLSNISNLKADYIKEQLELPNDSNGWEIFPNPSYIFIDNNVPIHVLADEATKKGIELFKDKRRKEQPEELQKRKGKISKYNIVSMIYKEAKGKDKLRIDEPLALLSLNEIPALLYQILEKGATPKDIELIIKNKLTERFEKIKNYDPETPAPASQISKRLRNNTTAKGQEALNAEKLSLLIEREIENTETKLSSIEEKRLKAKKEQRRNTPQRSIFSNSDLGRIAAWLADDIKRFMPAEQRKNWKGYQHSQLQQSLAYFEKRPQEAFLLLKEGWDTSDGSSYWNNWVMNSFLENNHFEKFYKNYLMKRVKYFSELAGNIKQHTHNTKFLRKFIKQQMPADLFPKRHYILKDLETEKNKVLSKPLVFSRGLFDNNPTFIKGVKVTENPELFAEWYSYGYKTEHVFQHFYGWERDYNELLDSELQKGNSFAKNSIYYNRESQLDLIKLKQDLKIKKIKIQDLFLKRIAEKLFENVFNYPTTLSLDEFYLTQEERAEKERIALAQSLREEGDNSPNIIKDDFIWSKTIAFRSKQIYEPAIKLKDIGKFNRFVLDDEESKASKLLSYDKNKIWNKEQLERELSIGENSYEVIRREKLFKEIQNLELQILSNWSWDGINHPREFEMEDQKNTRHPNFKMYLVNGILRKNINLYKEDEDFWLESLKENDFKTLPSEVLETKSEMVQLLFLVILIRNQFAHNQLPEIQFYNFIRKNYPEIQNNTVAELYLNLIKLAVQKLKDNS (SEQ ID NO: 35) SAMN054 SHM52812.1MNTRVTGMGVSYDHTKKEDKHFFGGFLNLAQDNITAVIKAFCI 44360_11366KFDKNPMSSVQFAESCFTDKDSDTDFQNKVRYVRTHLPVIGYL [Chryseo-NYGGDRNTFRQKLSTLLKAVDSLRNFYTHYYHSPLALSTELFEL bacteriumLDTVFASVAVEVKQHKMKDDKTRQLLSKSLAEELDIRYKQQLE carnipullorum]RLKELKEQGKNIDLRDEAGIRNGVLNAAFNHLIYKEGEIAKPTLSYSSFYYGADSAENGITISQSGLLFLLSMFLGKKEIEDLKSRIRGFKAKIVRDGEENISGLKFMATHWIFSYLSFKGMKQRLSTDFHEETLLIQIIDELSKVPDEVYHDFDTATREKFVEDINEYIREGNEDFSLGDSTIIHPVIRKRYENKFNYFAVRFLDEFIKFPSLRFQVHLGNFVHDRRIKDIHGTGFQTERVVKDRIKVFGKLSEISSLKTEYIEKELDLDSDTGWEIFPNPSYVFIDNNIPIYISTNKTFKNGSSEFIKLRRKEKPEEMKMRGEDKKEKRDIASMIGNAGSLNSKTPLAMLSLNEMPALLYEILVKKTTPEEIELIIKEKLDSHFENIKNYDPEKPLPASQISKRLRNNTTDKGKKVINPEKLIHLINKEIDATEAKFALLAKNRKELKEKFRGKPLRQTIFSNMELGREATWLADDIKRFMPDILRKNWKGYQHNQLQQSLAFFNSRPKEAFTILQDGWDFADGSSFWNGWIINSFVKNRSFEYFYEAYFEGRKEYFSSLAENIKQHTSNHRNLRRFIDQQMPKGLFENRHYLLENLETEKNKILSKPLVFPRGLFDTKPTFIKGIKVDEQPELFAEWYQYGYSTEHVFQNFYGWERDYNDLLESELEKDNDFSKNSIHYSRTSQLELIKLKQDLKIKKIKIQDLFLKLIAGHIFENIFKYPASFSLDELYLTQEERLNKEQEALIQSQRKEGDHSDNIIKDNFIGSKTVTYESKQISEPNVKLKDIGKFNRFLLDDKVKTLLSYNEDKVWNKNDLDLELSIGENSYEVIRREKLFKKIQNFELQTLTDWPWNGTDHPEEFGTTDNKGVNHPNFKMYVVNGILRKHTDWFKEGEDNWLENLNETHFKNLSFQELETKSKSIQTAFLIIMIRNQFAHNQLPAVQFFEFIQKKYPEIQGSTTSELYLNFINLAVVELLELL EK (SEQ ID NO: 36) SAMN054SIS70481.1 METQILGNGISYDHTKTEDKHFFGGFLNTAQNNIDLLIKAYISKF 21786_1011119ESSPRKLNSVQFPDVCFKKNDSDADFQHKLQFIRKHLPVIQYLK [Chryseo-YGGNREVLKEKFRLLLQAVDSLRNFYTHFYHKPIQLPNELLTLL bacteriumDTIFGEIGNEVRQNKMKDDKTRHLLKKNLSEELDFRYQEQLER ureilyticum]LRKLKSEGKKVDLRDTEAIRNGVLNAAFNHLIFKDAEDFKPTVSYSSYYYDSDTAENGISISQSGLLFLLSMFLGRREMEDLKSRVRGFKARIIKHEEQHVSGLKFMATHWVFSEFCFKGIKTRLNADYHEETLLIQLIDELSKVPDELYRSFDVATRERFIEDINEYIRDGKEDKSLIESKIVHPVIRKRYESKFNYFAIRFLDEFVNFPTLRFQVHAGNYVHDRRIKSIEGTGFKTERLVKDRIKVFGKLSTISSLKAEYLAKAVNITDDTGWELLPHPSYVFIDNNIPIHLTVDPSFKNGVKEYQEKRKLQKPEEMKNRQGGDKMHKPAISSKIGKSKDINPESPVALLSMNEIPALLYEILVKKASPEEVEAKIRQKLTAVFERIRDYDPKVPLPASQVSKRLRNNTDTLSYNKEKLVELANKEVEQTERKLALITKNRRECREKVKGKFKRQKVFKNAELGTEATWLANDIKRFMPEEQKKNWKGYQHSQLQQSLAFFESRPGEARSLLQAGWDFSDGSSFWNGWVMNSFARDNTFDGFYESYLNGRMKYFLRLADNIAQQSSTNKLISNFIKQQMPKGLFDRRLYMLEDLATEKNKILSKPLIFPRGIFDDKPTFKKGVQVSEEPEAFADWYSYGYDVKHKFQEFYAWDRDYEELLREELEKDTAFTKNSIHYSRESQIELLAKKQDLKVKKVRIQDLYLKLMAEFLFENVFGHELALPLDQFYLTQEERLKQEQEAIVQSQRPKGDDSPNIVKENFIWSKTIPFKSGRVFEPNVKLKDIGKFRNLLTDEKVDILLSYNNTEIGKQVIENELIIGAGSYEFIRREQLFKEIQQMKRLSLRSVRGMGVPIRLNLK (SEQ ID NO: 37) Reichen- WP_073124441.1MKTNPLIASSGEKPNYKKFNTESDKSFKKIFQNKGSIAPIAEKAC bachiellaKNFEIKSKSPVNRDGRLHYFSVGHAFKNIDSKNVFRYELDESQM agariperforansDMKPTQFLALQKEFFDFQGALNGLLKHIRNVNSHYVHTFEKLEIQSINQKLITFLIEAFELAVIHSYLNEEELSYEAYKDDPQSGQKLVQFLCDKFYPNKEHEVEERKTILAKNKRQALEHLLFIEVTSDIDWKLFEKHKVFTISNGKYLSFHACLFLLSLFLYKSEANQLISKIKGFKRNDDNQYRSKRQIFTFFSKKFTSQDVNSEEQHLVKFRDVIQYLNHYPSAWNKHLELKSGYPQMTDKLMRYIVEAEIYRSFPDQTDNHRFLLFAIREFFGQSCLDTWTGNTPINFSNQEQKGFSYEINTSAEIKDIETKLKALVLKGPLNFKEKKEQNRLEKDLRREKKEQPTNRVKEKLLTRIQHNMLYVSYGRNQDRFMDFAARFLAETDYFGKDAKFKMYQFYTSDEQRDHLKEQKKELPKKEFEKLKYHQSKLVDYFTYAEQQARYPDWDTPFVVENNAIQIKVTLFNGAKKIVSVQRNLMLYLLEDALYSEKRENAGKGLISGYFVHHQKELKDQLDILEKETEISREQKREFKKLLPKRLLHRYSPAQINDTTEWNPMEVILEEAKAQEQRYQLLLEKAILHQTEEDFLKRNKGKQFKLRFVRKAWHLMYLKELYMNKVAEHGHHKSFHITKEEFNDFCRWMFAFDEVPKYKEYLCDYFSQKGFFNNAEFKDLIESSTSLNDLYEKTKQRFEGWSKDLTKQSDENKYLLANYESMLKDDMLYVNISHFISYLESKGKINRNAHGHIAYKALNNVPHLIEEYYYKDRLAPEEYKSHGKLYNKLKTVKLEDALLYEMAMHYLSLEPALVPKVKTKVKDILSSNIAFDIKDAAGHHLYHLLIPFHKIDSFVALINHQSQQEKDPDKTSFLAKIQPYLEKVKNSKDLKAVYHYYKDTPHTLRYEDLNMIHSHIVSQSVQFTKVALKLEEYFIAKKSITLQIARQISYSEIADLSNYFTDEVRNTAFHFDVPETAYSMILQGIESEFLDREIKPQKPKSLSELSTQQVSVCTAFLETLHNNLFDRKDDKKERLSKARERYFEQIN (SEQ ID NO: 38)

TABLE 1B Bergeyella  1 MENKTSLGNNIYYNPFKPQDKSYFAGYFNAAMENTDSVFRELGzoohelcum KRLKGKEYTSENFFDAIFKENISLVEYERYVKLLSDYFPMARLLDKKEVPIKERKENFKKNFKGIIKAVRDLRNFYTHKEHGEVEITDEIFGVLDEMLKSTVLTVKKKKVKTDKTKEILKKSIEKQLDILCQKKLEYLRDTARKIEEKRRNQRERGEKELVAPFKYSDKRDDLIAAIYNDAFDVYIDKKKDSLKESSKAKYNTKSDPQQEEGDLKIPISKNGVVFLLSLFLTKQEIHAFKSKIAGFKATVIDEATVSEATVSHGKNSICFMATHEIFSHLAYKKLKRKVRTAEINYGEAENAEQLSVYAKETLMMQMLDELSKVPDVVYQNLSEDVQKTFIEDWNEYLKENNGDVGTMEEEQVIHPVIRKRYEDKFNYFAIRFLDEFAQFPTLRFQVHLGNYLHDSRPKENLISDRRIKEKITVFGRLSELEHKKALFIKNTETNEDREHYWEIFPNPNYDFPKENISVNDKDFPIAGSILDREKQPVAGKIGIKVKLLNQQYVSEVDKAVKAHQLKQRKASKPSIQNIIEEIVPINESNPKEAIVFGGQPTAYLSMNDIHSILYEFFDKWEKKKEKLEKKGEKELRKEIGKELEKKIVGKIQAQIQQIIDKDTNAKILKPYQDGNSTAIDKEKLIKDLKQEQNILQKLKDEQTVREKEYNDFIAYQDKNREINKVRDRNHKQYLKDNLKRKYPEAPARKEVLYYREKGKVAVWLANDIKRFMPTDFKNEWKGEQHSLLQKSLAYYEQCKEELKNLLPEKVFQHLPFKLGGYFQQKYLYQFYTCYLDKRLEYISGLVQQAENFKSENKVFKKVENECFKFLKKQNYTHKELDARVQSILGYPIFLERGFMDEKPTIIKGKTFKGNEALFADWFRYYKEYQNFQTFYDTENYPLVELEKKQADRKRKTKIYQQKKNDVFTLLMAKHIFKSVFKQDSIDQFSLEDLYQSREERLGNQERARQTGERNTNYIWNKTVDLKLCDGKITVENVKLKNVGDFIKYEYDQRVQAFLKYEENIEWQAFLIKESKEEENYPYVVEREIEQYEKVRREELLKEVHLIEEYILEKVKDKEILKKGDNQNFKYYILNGLLKQLKNEDVESYKVFNLNTEPEDVNINQLKQEATDLEQKAFVLTYIRNKFAHNQLPKKEFWDYCQEKYGKIEKEKTYAEYFAEVFKKEKE ALIK (SEQ ID NO: 39)Prevotella  2 MEDDKKTTDSIRYELKDKHFWAAFLNLARHNVYITVNHINKIL intermediaEEGEINRDGYETTLKNTWNEIKDINKKDRLSKLIIKHFPFLEAATYRLNPTDTTKQKEEKQAEAQSLESLRKSFFVFIYKLRDLRNHYSHYKHSKSLERPKFEEGLLEKMYNIFNASIRLVKEDYQYNKDINPDEDFKHLDRTEEEFNYYFTKDNEGNITESGLLFFVSLFLEKKDAIWMQQKLRGFKDNRENKKKMTNEVFCRSRMLLPKLRLQSTQTQDWILLDMLNELIRCPKSLYERLREEDREKFRVPIEIADEDYDAEQEPFKNTLVRHQDRFPYFALRYFDYNEIFTNLRFQIDLGTYHFSIYKKQIGDYKESHHLTHKLYGFERIQEFTKQNRPDEWRKFVKTFNSFETSKEPYIPETTPHYHLENQKIGIRFRNDNDKIWPSLKTNSEKNEKSKYKLDKSFQAEAFLSVHELLPMMFYYLLLKTENTDNDNEIETKKKENKNDKQEKHKIEEIIENKITEIYALYDTFANGEIKSIDELEEYCKGKDIEIGHLPKQMIAILKDEHKVMATEAERKQEEMLVDVQKSLESLDNQINEEIENVERKNSSLKSGKIASWLVNDMMRFQPVQKDNEGKPLNNSKANSTEYQLLQRTLAFFGSEHERLAPYFKQTKLIESSNPHPFLKDTEWEKCNNILSFYRSYLEAKKNFLESLKPEDWEKNQYFLKLKEPKTKPKTLVQGWKNGFNLPRGIFTEPIRKWFMKHRENITVAELKRVGLVAKVIPLFFSEEYKDSVQPFYNYHFNVGNINKPDEKNFLNCEERRELLRKKKDEFKKMTDKEKEENPSYLEFKSWNKFERELRLVRNQDIVTWLLCMELFNKKKIKELNVEKIYLKNINTNTTKKEKNTEEKNGEEKNIKEKNNILNRIMPMRLPIKVYGRENFSKNKKKKIRRNTFFTVYIEEKGTKLLKQGNFKALERDRRLGGLFSFVKTPSKAESKSNTISKLRVEYELGEYQKARIEIIKDMLALEKTLIDKYNSLDTDNFNKMLTDWLELKGEPDKASFQNDVDLLIAVRNAFSHNQYPMRNRIAFANINPFSLSSANTSEEKGLGIANQLKDKTHKTIEKIIEIEKPIETKE (SEQ ID NO: 40) Prevotella  3MQKQDKLFVDRKKNAIFAFPKYITIMENKEKPEPIYYELTDKHF buccaeWAAFLNLARHNVYTTINHINRRLEIAELKDDGYMMGIKGSWNEQAKKLDKKVRLRDLIMKHFPFLEAAAYEMTNSKSPNNKEQREKEQSEALSLNNLKNVLFIFLEKLQVLRNYYSHYKYSEESPKPIFETSLLKNMYKVFDANVRLVKRDYMHHENIDMQRDFTHLNRKKQVGRTKNIIDSPNFHYHFADKEGNMTIAGLLFFVSLFLDKKDAIWMQKKLKGFKDGRNLREQMTNEVFCRSRISLPKLKLENVQTKDWMQLDMLNELVRCPKSLYERLREKDRESFKVPFDIFSDDYNAEEEPFKNTLVRHQDRFPYFVLRYFDLNEIFEQLRFQIDLGTYHFSIYNKRIGDEDEVRHLTHHLYGFARIQDFAPQNQPEEWRKLVKDLDHFETSQEPYISKTAPHYHLENEKIGIKFCSAHNNLFPSLQTDKTCNGRSKFNLGTQFTAEAFLSVHELLPMMFYYLLLTKDYSRKESADKVEGIIRKEISNIYAIYDAFANNEINSIADLTRRLQNTNILQGHLPKQMISILKGRQKDMGKEAERKIGEMIDDTQRRLDLLCKQTNQKIRIGKRNAGLLKSGKIADWLVNDMMRFQPVQKDQNNIPINNSKANSTEYRMLQRALALFGSENFRLKAYFNQMNLVGNDNPHPFLAETQWEHQTNILSFYRNYLEARKKYLKGLKPQNWKQYQHFLILKVQKTNRNTLVTGWKNSFNLPRGIFTQPIREWFEKHNNSKRIYDQILSFDRVGFVAKAIPLYFAEEYKDNVQPFYDYPFNIGNRLKPKKRQFLDKKERVELWQKNKELFKNYPSEKKKTDLAYLDFLSWKKFERELRLIKNQDIVTWLMFKELFNMATVEGLKIGEIHLRDIDTNTANEESNNILNRIMPMKLPVKTYETDNKGNILKERPLATFYIEETETKVLKQGNFKALVKDRRLNGLFSFAETTDLNLEEHPISKLSVDLELIKYQTTRISIFEMTLGLEKKLIDKYSTLPTDSFRNMLERWLQCKANRPELKNYVNSLIAVRNAFSHNQYPMYDATLFAEVKKFTLFPSVDTKKIELNIAPQLLEIVGKAIKEIEKSENKN (SEQ ID NO: 41) Porphyromonas  4MNTVPASENKGQSRTVEDDPQYFGLYLNLARENLIEVESHVRIK gingivalisFGKKKLNEESLKQSLLCDHLLSVDRWTKVYGHSRRYLPFLHYFDPDSQIEKDHDSKTGVDPDSAQRLIRELYSLLDFLRNDFSHNRLDGTTFEHLEVSPDISSFITGTYSLACGRAQSRFAVFFKPDDFVLAKNRKEQLISVADGKECLTVSGFAFFICLFLDREQASGMLSRIRGFKRTDENWARAVHETFCDLCIRHPHDRLESSNTKEALLLDMLNELNRCPRILYDMLPEEERAQFLPALDENSMNNLSENSLDEESRLLWDGSSDWAEALTKRIRHQDRFPYLMLRFIEEMDLLKGIRFRVDLGEIELDSYSKKVGRNGEYDRTITDHALAFGKLSDFQNEEEVSRMISGEASYPVRFSLFAPRYAIYDNKIGYCHTSDPVYPKSKTGEKRALSNPQSMGFISVHDLRKLLLMELLCEGSFSRMQSDFLRKANRILDETAEGKLQFSALFPEMRHRFIPPQNPKSKDRREKAETTLEKYKQEIKGRKDKLNSQLLSAFDMDQRQLPSRLLDEWMNIRPASHSVKLRTYVKQLNEDCRLRLRKFRKDGDGKARAIPLVGEMATFLSQDIVRMIISEETKKLITSAYYNEMQRSLAQYAGEENRRQFRAIVAELRLLDPSSGHPFLSATMETAHRYTEGFYKCYLEKKREWLAKIFYRPEQDENTKRRISVFFVPDGEARKLLPLLIRRRMKEQNDLQDWIRNKQAHPIDLPSHLFDSKVMELLKVKDGKKKWNEAFKDWWSTKYPDGMQPFYGLRRELNIHGKSVSYIPSDGKKFADCYTHLMEKTVRDKKRELRTAGKPVPPDLAADIKRSFHRAVNEREFMLRLVQEDDRLMLMAINKMMTDREEDILPGLKNIDSILDEENQFSLAVHAKVLEKEGEGGDNSLSLVPATIEIKSKRKDWSKYIRYRYDRRVPGLMSHFPEHKATLDEVKTLLGEYDRCRIKIFDWAFALEGAIMSDRDLKPYLHESSSREGKSGEHSTLVKMLVEKKGCLTPDESQYLILIRNKAAHNQFPCAAEMPLIYRDVSAKVGSIEGSSAKDLPEGSSLVDSLWKKYEMIIRKILPILDPENRFFGKLLNNMSQPINDL (SEQ ID NO: 42) Bacteroides 5 MESIKNSQKSTGKTLQKDPPYFGLYLNMALLNVRKVENHIRKW pyogenesLGDVALLPEKSGFHSLLTTDNLSSAKWTRFYYKSRKFLPFLEMFDSDKKSYENRRETAECLDTIDRQKISSLLKEVYGKLQDIRNAFSHYHIDDQSVKHTALIISSEMHRFIENAYSFALQKTRARFTGVFVETDFLQAEEKGDNKKFFAIGGNEGIKLKDNALIFLICLFLDREEAFKFLSRATGFKSTKEKGFLAVRETFCALCCRQPHERLLSVNPREALLMDMLNELNRCPDILFEMLDEKDQKSFLPLLGEEEQAHILENSLNDELCEAIDDPFEMIASLSKRVRYKNRFPYLMLRYIEEKNLLPFIRFRIDLGCLELASYPKKMGEENNYERSVTDHAMAFGRLTDFHNEDAVLQQITKGITDEVRFSLYAPRYAIYNNKIGFVRTSGSDKISFPTLKKKGGEGHCVAYTLQNTKSFGFISIYDLRKILLLSFLDKDKAKNIVSGLLEQCEKHWKDLSENLFDAIRTELQKEFPVPLIRYTLPRSKGGKLVSSKLADKQEKYESEFERRKEKLTEILSEKDFDLSQIPRRMIDEWLNVLPTSREKKLKGYVETLKLDCRERLRVFEKREKGEHPLPPRIGEMATDLAKDIIRMVIDQGVKQRITSAYYSEIQRCLAQYAGDDNRRHLDSIIRELRLKDTKNGHPFLGKVLRPGLGHTEKLYQRYFEEKKEWLEATFYPAASPKRVPRFVNPPTGKQKELPLIIRNLMKERPEWRDWKQRKNSHPIDLPSQLFENEICRLLKDKIGKEPSGKLKWNEMFKLYWDKEFPNGMQRFYRCKRRVEVFDKVVEYEYSEEGGNYKKYYEALIDEVVRQKISSSKEKSKLQVEDLTLSVRRVFKRAINEKEYQLRLLCEDDRLLFMAVRDLYDWKEAQLDLDKIDNMLGEPVSVSQVIQLEGGQPDAVIKAECKLKDVSKLMRYCYDGRVKGLMPYFANHEATQEQVEMELRHYEDHRRRVFNWVFALEKSVLKNEKLRRFYEESQGGCEHRRCIDALRKASLVSEEEYEFLVHIRNKSAHNQFPDLEIGKLPPNVTSGFCECIWSKYKAIICRIIPFIDPERRFFGKLLEQK (SEQ ID NO: 43) Alistipes  6MSNEIGAFREHQFAYAPGNEKQEEATFATYFNLALSNVEGMMF sp.GEVESNPDKIEKSLDTLPPAILRQIASFIWLSKEDHPDKAYSTEE ZOR0009VKVIVTDLVRRLCFYRNYFSHCFYLDTQYFYSDELVDTTAIGEKLPYNFHHFITNRLFRYSLPEITLFRWNEGERKYEILRDGLIFFCCLFLKRGQAERFLNELRFFKRTDEEGRIKRTIFTKYCTRESHKHIGIEEQDFLIFQDIIGDLNRVPKVCDGVVDLSKENERYIKNRETSNESDENKARYRLLIREKDKFPYYLMRYIVDFGVLPCITFKQNDYSTKEGRGQFHYQDAAVAQEERCYNFVVRNGNVYYSYMPQAQNVVRISELQGTISVEELRNMVYASINGKDVNKSVEQYLYHLHLLYEKILTISGQTIKEGRVDVEDYRPLLDKLLLRPASNGEELRRELRKLLPKRVCDLLSNRFDCSEGVSAVEKRLKAILLRHEQLLLSQNPALHIDKIKSVIDYLYLFFSDDEKFRQQPTEKAHRGLKDEEFQMYHYLVGDYDSHPLALWKELEASGRLKPEMRKLTSATSLHGLYMLCLKGTVEWCRKQLMSIGKGTAKVEAIADRVGLKLYDKLKEYTPEQLEREVKLVVMHGYAAAATPKPKAQAAIPSKLTELRFYSFLGKREMSFAAFIRQDKKAQKLWLRNFYTVENIKTLQKRQAAADAACKKLYNLVGEVERVHTNDKVLVLVAQRYRERLLNVGSKCAVTLDNPERQQKLADVYEVQNAWLSIRFDDLDFTLTHVNLSNLRKAYNLIPRKHILAFKEYLDNRVKQKLCEECRNVRRKEDLCTCCSPRYSNLTSWLKENHSESSIEREAATMMLLDVERKLLSFLLDERRKAIIEYGKFIPFSALVKECRLADAGLCGIRNDVLHDNVISYADAIGKLSAYFPKEASEAVEYIRRTKEVREQRREELMANSSQ (SEQ ID NO: 44) Prevotella  7aMSKECKKQRQEKKRRLQKANFSISLTGKHVFGAYFNMARTNF sp.VKTINYILPIAGVRGNYSENQINKMLHALFLIQAGRNEELTTEQK MA2016QWEKKLRLNPEQQTKFQKLLFKHFPVLGPMMADVADHKAYLNKKKSTVQTEDETFAMLKGVSLADCLDIICLMADTLTECRNFYTHKDPYNKPSQLADQYLHQEMIAKKLDKVVVASRRILKDREGLSVNEVEFLTGIDHLHQEVLKDEFGNAKVKDGKVMKTFVEYDDFYFKISGKRLVNGYTVTTKDDKPVNVNTMLPALSDFGLLYFCVLFLSKPYAKLFIDEVRLFEYSPFDDKENMIMSEMLSIYRIRTPRLHKIDSHDSKATLAMDIFGELRRCPMELYNLLDKNAGQPFFHDEVKHPNSHTPDVSKRLRYDDRFPTLALRYIDETELFKRIRFQLQLGSFRYKFYDKENCIDGRVRVRRIQKEINGYGRMQEVADKRMDKWGDLIQKREERSVKLEHEELYINLDQFLEDTADSTPYVTDRRPAYNIHANRIGLYWEDSQNPKQYKVFDENGMYIPELVVTEDKKAPIKMPAPRCALSVYDLPAMLFYEYLREQQDNEFPSAEQVIIEYEDDYRKFFKAVAEGKLKPFKRPKEFRDFLKKEYPKLRMADIPKKLQLFLCSHGLCYNNKPETVYERLDRLTLQHLEERELHIQNRLEHYQKDRDMIGNKDNQYGKKSFSDVRHGALARYLAQSMMEWQPTKLKDKEKGHDKLTGLNYNVLTAYLATYGHPQVPEEGFTPRTLEQVLINAHLIGGSNPHPFINKVLALGNRNIEELYLHYLEEELKHIRSRIQSLSSNPSDKALSALPFIHHDRMRYHERTSEEMMALAARYTTIQLPDGLFTPYILEILQKHYTENSDLQNALSQDVPVKLNPTCNAAYLITLFYQTVLKDNAQPFYLSDKTYTRNKDGEKAESFSFKRAYELFSVLNNNKKDTFPFEMIPLFLTSDEIQERLSAKLLDGDGNPVPEVGEKGKPATDSQGNTIWKRRIYSEVDDYAEKLTDRDMKISFKGEWEKLPRWKQDKIIKRRDETRRQMRDELLQRMPRYIRDIKDNERTLRRYKTQDMVLFLLAEKMFTNIISEQSSEFNWKQMRLSKVCNEAFLRQTLTFRVPVTVGETTIYVEQENMSLKNYGEFYRFLTDDRLMSLLNNIVETLKPNENGDLVIRHTDLMSELAAYDQYRSTIFMLIQSIENLIITNNAVLDDPDADGFWVREDLPKRNNFASLLELINQLNNVELTDDERKLLVAIRNAFSHNSYNIDFSLIKDVKHLPEVAKGILQHLQSMLGVEITK (SEQ ID NO: 45) Prevotella  7bMSKECKKQRQEKKRRLQKANFSISLTGKHVFGAYFNMARTNF sp.VKTINYILPIAGVRGNYSENQINKMLHALFLIQAGRNEELTTEQK MA2016QWEKKLRLNPEQQTKFQKLLFKHFPVLGPMMADVADHKAYLNKKKSTVQTEDETFAMLKGVSLADCLDIICLMADTLTECRNFYTHKDPYNKPSQLADQYLHQEMIAKKLDKVVVASRRILKDREGLSVNEVEFLTGIDHLHQEVLKDEFGNAKVKDGKVMKTFVEYDDFYFKISGKRLVNGYTVTTKDDKPVNVNTMLPALSDFGLLYFCVLFLSKPYAKLFIDEVRLFEYSPFDDKENMIMSEMLSIYRIRTPRLHKIDSHDSKATLAMDIFGELRRCPMELYNLLDKNAGQPFFHDEVKHPNSHTPDVSKRLRYDDRFPTLALRYIDETELFKRIRFQLQLGSFRYKFYDKENCIDGRVRVRRIQKEINGYGRMQEVADKRMDKWGDLIQKREERSVKLEHEELYINLDQFLEDTADSTPYVTDRRPAYNIHANRIGLYWEDSQNPKQYKVFDENGMYIPELVVTEDKKAPIKMPAPRCALSVYDLPAMLFYEYLREQQDNEFPSAEQVIIEYEDDYRKFFKAVAEGKLKPFKRPKEFRDFLKKEYPKLRMADIPKKLQLFLCSHGLCYNNKPETVYERLDRLTLQHLEERELHIQNRLEHYQKDRDMIGNKDNQYGKKSFSDVRHGALARYLAQSMMEWQPTKLKDKEKGHDKLTGLNYNVLTAYLATYGHPQVPEEGFTPRTLEQVLINAHLIGGSNPHPFINKVLALGNRNIEELYLHYLEEELKHIRSRIQSLSSNPSDKALSALPFIHHDRMRYHERTSEEMMALAARYTTIQLPDGLFTPYILEILQKHYTENSDLQNALSQDVPVKLNPTCNAAYLITLFYQTVLKDNAQPFYLSDKTYTRNKDGEKAESFSFKRAYELFSVLNNNKKDTFPFEMIPLFLTSDEIQERLSAKLLDGDGNPVPEVGEKGKPATDSQGNTIWKRRIYSEVDDYAEKLTDRDMKISFKGEWEKLPRWKQDKIIKRRDETRRQMRDELLQRMPRYIRDIKDNERTLRRYKTQDMVLFLLAEKMFTNIISEQSSEFNWKQMRLSKVCNEAFLRQTLTFRVPVTVGETTIYVEQENMSLKNYGEFYRFLTDDRLMSLLNNIVETLKPNENGDLVIRHTDLMSELAAYDQYRSTIFMLIQSIENLIITNNAVLDDPDADGFWVREDLPKRNNFASLLELINQLNNVELTDDERKLLVAIRNAFSHNSYNIDFSLIKDVKHLPEVAKGILQHLQSMLGVEITK (SEQ ID NO: 46) Riemerella  8MEKPLLPNVYTLKHKFFWGAFLNIARHNAFITICHINEQLGLKT anatipestiferPSNDDKIVDVVCETWNNILNNDHDLLKKSQLTELILKHFPFLTAMCYHPPKKEGKKKGHQKEQQKEKESEAQSQAEALNPSKLIEALEILVNQLHSLRNYYSHYKHKKPDAEKDIFKHLYKAFDASLRMVKEDYKAHFTVNLTRDFAHLNRKGKNKQDNPDFNRYRFEKDGFFTESGLLFFTNLFLDKRDAYWMLKKVSGFKASHKQREKMTTEVFCRSRILLPKLRLESRYDHNQMLLDMLSELSRCPKLLYEKLSEENKKHFQVEADGFLDEIEEEQNPFKDTLIRHQDRFPYFALRYLDLNESFKSIRFQVDLGTYHYCIYDKKIGDEQEKRHLTRTLLSFGRLQDFTEINRPQEWKALTKDLDYKETSNQPFISKTTPHYHITDNKIGFRLGTSKELYPSLEIKDGANRIAKYPYNSGFVAHAFISVHELLPLMFYQHLTGKSEDLLKETVRHIQRIYKDFEEERINTIEDLEKANQGRLPLGAFPKQMLGLLQNKQPDLSEKAKIKIEKLIAETKLLSHRLNTKLKSSPKLGKRREKLIKTGVLADWLVKDFMRFQPVAYDAQNQPIKSSKANSTEFWFIRRALALYGGEKNRLEGYFKQTNLIGNTNPHPFLNKFNWKACRNLVDFYQQYLEQREKFLEAIKNQPWEPYQYCLLLKIPKENRKNLVKGWEQGGISLPRGLFTEAIRETLSEDLMLSKPIRKEIKKHGRVGFISRAITLYFKEKYQDKHQSFYNLSYKLEAKAPLLKREEHYEYWQQNKPQSPTESQRLELHTSDRWKDYLLYKRWQHLEKKLRLYRNQDVMLWLMTLELTKNHFKELNLNYHQLKLENLAVNVQEADAKLNPLNQTLPMVLPVKVYPATAFGEVQYHKTPIRTVYIREEHTKALKMGNFKALVKDRRLNGLFSFIKEENDTQKHPISQLRLRRELEIYQSLRVDAFKETLSLEEKLLNKHTSLSSLENEFRALLEEWKKEYAASSMVTDEHIAFIASVRNAFCHNQYPFYKEALHAPIPLFTVAQPTTEEKDGLGIAEALLKVLREYC EIVKSQI (SEQ ID NO: 47)Prevotella  9 MEDDKKTTGSISYELKDKHFWAAFLNLARHNVYITINHINKLLE aurantiacaIREIDNDEKVLDIKTLWQKGNKDLNQKARLRELMTKHFPFLETAIYTKNKEDKKEVKQEKQAEAQSLESLKDCLFLFLDKLQEARNYYSHYKYSEFSKEPEFEEGLLEKMYNIFGNNIQLVINDYQHNKDINPDEDFKHLDRKGQFKYSFADNEGNITESGLLFFVSLFLEKKDAIWMQQKLNGFKDNLENKKKMTHEVFCRSRILMPKLRLESTQTQDWILLDMLNELIRCPKSLYERLQGDDREKFKVPFDPADEDYNAEQEPFKNTLIRHQDRFPYFVLRYFDYNEIFKNLRFQIDLGTYHFSIYKKLIGGQKEDRHLTHKLYGFERIQEFAKQNRPDEWKAIVKDLDTYETSNKRYISETTPHYHLENQKIGIRFRNGNKEIWPSLKINDENNEKSKYKLDKQYQAEAFLSVHELLPMMFYYLLLKKEKPNNDEINASIVEGFIKREIRNIFKLYDAFANGEINNIDDLEKYCADKGIPKRHLPKQMVAILYDEHKDMVKEAKRKQKEMVKDTKKLLATLEKQTQKEKEDDGRNVKLLKSGEIARWLVNDMMRFQPVQKDNEGKPLNNSKANSTEYQMLQRSLALYNNEEKPTRYFRQVNLIESNNPHPFLKWTKWEECNNILTFYYSYLTKKIEFLNKLKPEDWKKNQYFLKLKEPKTNRETLVQGWKNGFNLPRGIFTEPIREWFKRHQNNSKEYEKVEALDRVGLVTKVIPLFFKEEYFKDKEENFKEDTQKEINDCVQPFYNFPYNVGNIHKPKEKDFLHREERIELWDKKKDKFKGYKEKIKSKKLTEKDKEEFRSYLEFQSWNKFERELRLVRNQDIVTWLLCKELIDKLKIDELNIEELKKLRLNNIDTDTAKKEKNNILNRVMPMELPVTVYEIDDSHKIVKDKPLHTIYIKEAETKLLKQGNFKALVKDRRLNGLFSFVKTNSEAESKRNPISKLRVEYELGEYQEARIEIIQDMLALEEKLINKYKDLPTNKFSEMLNSWLEGKDEADKARFQNDVDFLIAVRNAFSHNQYPMHNKIEFANIKPFSLYTANNSEEKGLGIANQLKDKTKETTDKIKKIEKPIETKE (SEQ ID NO: 48) Prevotella 10MEDKPFWAAFFNLARHNVYLTVNHINKLLDLEKLYDEGKHKEI saccharolyticaFEREDIFNISDDVMNDANSNGKKRKLDIKKIWDDLDTDLTRKYQLRELILKHFPFIQPAIIGAQTKERTTIDKDKRSTSTSNDSLKQTGEGDINDLLSLSNVKSMFFRLLQILEQLRNYYSHVKHSKSATMPNFDEDLLNWMRYIFIDSVNKVKEDYSSNSVIDPNTSFSHLIYKDEQGKIKPCRYPFTSKDGSINAFGLLFFVSLFLEKQDSIWMQKKIPGFKKASENYMKMTNEVFCRNHILLPKIRLETVYDKDWMLLDMLNEVVRCPLSLYKRLTPAAQNKFKVPEKSSDNANRQEDDNPFSRILVRHQNRFPYFVLRFFDLNEVFTTLRFQINLGCYHFAICKKQIGDKKEVHHLIRTLYGFSRLQNFTQNTRPEEWNTLVKTTEPSSGNDGKTVQGVPLPYISYTIPHYQIENEKIGIKIFDGDTAVDTDIWPSVSTEKQLNKPDKYTLTPGFKADVFLSVHELLPMMFYYQLLLCEGMLKTDAGNAVEKVLIDTRNAIFNLYDAFVQEKINTITDLENYLQDKPILIGHLPKQMIDLLKGHQRDMLKAVEQKKAMLIKDTERRLKLLDKQLKQETDVAAKNTGTLLKNGQIADWLVNDMMRFQPVKRDKEGNPINCSKANSTEYQMLQRAFAFYATDSCRLSRYFTQLHLIHSDNSHLFLSRFEYDKQPNLIAFYAAYLKAKLEFLNELQPQNWASDNYFLLLRAPKNDRQKLAEGWKNGFNLPRGLFTEKIKTWFNEHKTIVDISDCDIFKNRVGQVARLIPVFFDKKFKDHSQPFYRYDFNVGNVSKPTEANYLSKGKREELFKSYQNKFKNNIPAEKTKEYREYKNFSLWKKFERELRLIKNQDILIWLMCKNLFDEKIKPKKDILEPRIAVSYIKLDSLQTNTSTAGSLNALAKVVPMTLAIHIDSPKPKGKAGNNEKENKEFTVYIKEEGTKLLKWGNFKTLLADRRIKGLFSYIEHDDIDLKQHPLTKRRVDLELDLYQTCRIDIFQQTLGLEAQLLDKYSDLNTDNFYQMLIGWRKKEGIPRNIKEDTDFLKDVRNAFSHNQYPDSKKIAFRRIRKENPKELILEEEEGLGIATQMYKEVEKVVNRIKRIELFD (SEQ ID NO: 49) HMPREF9 11MKDILTTDTTEKQNRFYSHKIADKYFFGGYFNLASNNIYEVFEE 712_03108VNKRNTFGKLAKRDNGNLKNYIIHVFKDELSISDFEKRVAIFAS [MyroidesYFPILETVDKKSIKERNRTIDLTLSQRIRQFREMLISLVTAVDQLR odoratimimusNFYTHYHHSDIVIENKVLDFLNSSFVSTALHVKDKYLKTDKTKE CCUGFLKETIAAELDILIEAYKKKQIEKKNTRFKANKREDILNAIYNEA 10230]FWSFINDKDKDKDKETVVAKGADAYFEKNHHKSNDPDFALNISEKGIVYLLSFFLTNKEMDSLKANLTGFKGKVDRESGNSIKYMATQRIYSFHTYRGLKQKIRTSEEGVKETLLMQMIDELSKVPNVVYQHLSTTQQNSFIEDWNEYYKDYEDDVETDDLSRVIHPVIRKRYEDRFNYFAIRFLDEFFDFPTLRFQVHLGDYVHDRRTKQLGKVESDRIIKEKVTVFARLKDINSAKASYFHSLEEQDKEELDNKWTLFPNPSYDFPKEHTLQHQGEQKNAGKIGIYVKLRDTQYKEKAALEEARKSLNPKERSATKASKYDIITQIIEANDNVKSEKPLVFTGQPIAYLSMNDIHSMLFSLLTDNAELKKTPEEVEAKLIDQIGKQINEILSKDTDTKILKKYKDNDLKETDTDKITRDLARDKEEIEKLILEQKQRADDYNYTSSTKFNIDKSRKRKHLLFNAEKGKIGVWLANDIKRFMFKESKSKWKGYQHTELQKLFAYFDTSKSDLELILSNMVMVKDYPIELIDLVKKSRTLVDFLNKYLEARLEYIENVITRVKNSIGTPQFKTVRKECFTFLKKSNYTVVSLDKQVERILSMPLFIERGFMDDKPTMLEGKSYKQHKEKFADWFVHYKENSNYQNFYDTEVYEITTEDKREKAKVTKKIKQQQKNDVFTLMMVNYMLEEVLKLSSNDRLSLNELYQTKEERIVNKQVAKDTQERNKNYIWNKVVDLQLCDGLVHIDNVKLKDIGNFRKYENDSRVKEFLTYQSDIVWSAYLSNEVDSNKLYVIERQLDNYESIRSKELLKEVQEIECSVYNQVANKESLKQSGNENFKQYVLQGLLPIGMDVREMLILSTDVKFKKEEIIQLGQAGEVEQDLYSLIYIRNKFAHNQLPIKEFFDFCENNYRSISDNEYYAEYYMEIFRSIKEKYAN (SEQ ID NO: 50) Capnocyto 13MKNIQRLGKGNEFSPFKKEDKFYFGGFLNLANNNIEDFFKEIITR phagaFGIVITDENKKPKETFGEKILNEIFKKDISIVDYEKWVNIFADYFP canimorsusFTKYLSLYLEEMQFKNRVICFRDVMKELLKTVEALRNFYTHYDHEPIKIEDRVFYFLDKVLLDVSLTVKNKYLKTDKTKEFLNQHIGEELKELCKQRKDYLVGKGKRIDKESEIINGIYNNAFKDFICKREKQDDKENHNSVEKILCNKEPQNKKQKSSATVWELCSKSSSKYTEKSFPNRENDKHCLEVPISQKGIVFLLSFFLNKGEIYALTSNIKGFKAKITKEEPVTYDKNSIRYMATHRMFSFLAYKGLKRKIRTSEINYNEDGQASSTYEKETLMLQMLDELNKVPDVVYQNLSEDVQKTFIEDWNEYLKENNGDVGTMEEEQVIHPVIRKRYEDKFNYFAIRFLDEFAQFPTLRFQVHLGNYLCDKRTKQICDTTTEREVKKKITVFGRLSELENKKAIFLNEREEIKGWEVFPNPSYDFPKENISVNYKDFPIVGSILDREKQPVSNKIGIRVKIADELQREIDKAIKEKKLRNPKNRKANQDEKQKERLVNEIVSTNSNEQGEPVVFIGQPTAYLSMNDIHSVLYEFLINKISGEALETKIVEKIETQIKQIIGKDATTKILKPYTNANSNSINREKLLRDLEQEQQILKTLLEEQQQREKDKKDKKSKRKHELYPSEKGKVAVWLANDIKRFMPKAFKEQWRGYHHSLLQKYLAYYEQSKEELKNLLPKEVFKHFPFKLKGYFQQQYLNQFYTDYLKRRLSYVNELLLNIQNFKNDKDALKATEKECFKFFRKQNYIINPINIQIQSILVYPIFLKRGFLDEKPTMIDREKFKENKDTELADWFMHYKNYKEDNYQKFYAYPLEKVEEKEKFKRNKQINKQKKNDVYTLMMVEYIIQKIFGDKFVEENPLVLKGIFQSKAERQQNNTHAATTQERNLNGILNQPKDIKIQGKITVKGVKLKDIGNFRKYEIDQRVNTFLDYEPRKEWMAYLPNDWKEKEKQGQLPPNNVIDRQISKYETVRSKILLKDVQELEKIISDEIKEEHRHDLKQGKYYNFKYYILNGLLRQLKNENVENYKVFKLNTNPEKVNITQLKQEATDLEQKAFVLTYIRNKFAHNQLPKKEFWDYCQEKYGKIEKEKTYAEYFAE VFKREKEALIK (SEQ ID NO: 51)Porphyromonas 14 MTEQSERPYNGTYYTLEDKHFWAAFLNLARHNAYITLTHIDRQ gulaeLAYSKADITNDQDVLSFKALWKNFDNDLERKSRLRSLILKHFSFLEGAAYGKKLFESKSSGNKSSKNKELTKKEKEELQANALSLDNLKSILFDFLQKLKDFRNYYSHYRHSGSSELPLFDGNMLQRLYNVFDVSVQRVKIDHEHNDEVDPHYHFNHLVRKGKKDRYGHNDNPSFKHHFVDGEGMVTEAGLLFFVSLFLEKRDAIWMQKKIRGFKGGTETYQQMTNEVFCRSRISLPKLKLESLRMDDWMLLDMLNELVRCPKPLYDRLREDDRACFRVPVDILPDEDDTDGGGEDPFKNTLVRHQDRFPYFALRYFDLKKVFTSLRFHIDLGTYHFAIYKKMIGEQPEDRHLTRNLYGFGRIQDFAEEHRPEEWKRLVRDLDYFETGDKPYISQTSPHYHIEKGKIGLRFMPEGQHLWPSPEVGTTRTGRSKYAQDKRLTAEAFLSVHELMPMMFYYFLLREKYSEEVSAERVQGRIKRVIEDVYAVYDAFARDEINTRDELDACLADKGIRRGHLPRQMIAILSQEHKDMEEKIRKKLQEMMADTDHRLDMLDRQTDRKIRIGRKNAGLPKSGVIADWLVRDMMRFQPVAKDASGKPLNNSKANSTEYRMLQRALALFGGEKERLTPYFRQMNLTGGNNPHPFLHETRWESHTNILSFYRSYLRARKAFLERIGRSDRVENRPFLLLKEPKTDRQTLVAGWKGEFHLPRGIFTEAVRDCLIEMGHDEVASYKEVGFMAKAVPLYFERACEDRVQPFYDSPFNVGNSLKPKKGRFLSKEERAEEWERGKERFRDLEAWSYSAARRIEDAFAGIEYASPGNKKKIEQLLRDLSLWEAFESKLKVRADRINLAKLKKEILEAQEHPYHDFKSWQKFERELRLVKNQDIITWMMCRDLMEENKVEGLDTGTLYLKDIRPNVQEQGSLNVLNRVKPMRLPVVVYRADSRGHVHKEEAPLATVYIEERDTKLLKQGNFKSFVKDRRLNGLFSFVDTGGLAMEQYPISKLRVEYELAKYQTARVCVFELTLRLEESLLTRYPHLPDESFREMLESWSDPLLAKWPELHGKVRLLIAVRNAFSHNQYPMYDEAVFSSIRKYDPSSPDAIEERMGLNIAHRLSEEVKQAK ETVERIIQA (SEQ ID NO: 52)Prevotella 15 MNIPALVENQKKYFGTYSVMAMLNAQTVLDHIQKVADIEGEQ sp. P5-125NENNENLWFHPVMSHLYNAKNGYDKQPEKTMFIIERLQSYFPFLKIMAENQREYSNGKYKQNRVEVNSNDIFEVLKRAFGVLKMYRDLTNHYKTYEEKLNDGCEFLTSTEQPLSGMINNYYTVALRNMNERYGYKTEDLAFIQDKRFKFVKDAYGKKKSQVNTGFFLSLQDYNGDTQKKLHLSGVGIALLICLFLDKQYINIFLSRLPIFSSYNAQSEERRIIIRSFGINSIKLPKDRIHSEKSNKSVAMDMLNEVKRCPDELFTTLSAEKQSRFRIISDDHNEVLMKRSSDRFVPLLLQYIDYGKLFDHIRFHVNMGKLRYLLKADKTCIDGQTRVRVIEQPLNGFGRLEEAETMRKQENGTFGNSGIRIRDFENMKRDDANPANYPYIVDTYTHYILENNKVEMFINDKEDSAPLLPVIEDDRYVVKTIPSCRMSTLEIPAMAFHMFLFGSKKTEKLIVDVHNRYKRLFQAMQKEEVTAENIASFGIAESDLPQKILDLISGNAHGKDVDAFIRLTVDDMLTDTERRIKRFKDDRKSIRSADNKMGKRGFKQISTGKLADFLAKDIVLFQPSVNDGENKITGLNYRIMQSAIAVYDSGDDYEAKQQFKLMFEKARLIGKGTTEPHPFLYKVFARSIPANAVEFYERYLIERKFYLTGLSNEIKKGNRVDVPFIRRDQNKWKTPAMKTLGRIYSEDLPVELPRQMFDNEIKSHLKSLPQMEGIDENNANVTYLIAEYMKRVLDDDFQTFYQWNRNYRYMDMLKGEYDRKGSLQHCFTSVEEREGLWKERASRTERYRKQASNKIRSNRQMRNASSEEIETILDKRLSNSRNEYQKSEKVIRRYRVQDALLFLLAKKTLTELADFDGEREKLKEIMPDAEKGILSEIMPMSFTFEKGGKKYTITSEGMKLKNYGDFFVLASDKRIGNLLELVGSDIVSKEDIMEEFNKYDQCRPEISSIVENLEKWAFDTYPELSARVDREEKVDFKSILKILLNNKNINKEQSDILRKIRNAFDHNNYPDKGVVEIKALPEIAMSIKKAFGEYAIMK (SEQ ID NO: 53) Flavobacterium16 MENLNKILDKENEICISKIFNTKGIAAPITEKALDNIKSKQKNDL branchiophilumNKEARLHYFSIGHSFKQIDTKKVFDYVLIEELKDEKPLKFITLQKDFFTKEFSIKLQKLINSIRNINNHYVHNFNDINLNKIDSNVFHFLKESFELAIIEKYYKVNKKYPLDNEIVLFLKELFIKDENTALLNYFTNLSKDEAIEYILTFTITENKIWNINNEHNILNIEKGKYLTFEAMLFLITIFLYKNEANHLLPKLYDFKNNKSKQELFTFFSKKFTSQDIDAEEGHLIKFRDMIQYLNHYPTAWNNDLKLESENKNKIMTTKLIDSIIEFELNSNYPSFATDIQFKKEAKAFLFASNKKRNQTSFSNKSYNEEIRHNPHIKQYRDEIASALTPISFNVKEDKFKIFVKKHVLEEYFPNSIGYEKFLEYNDFTEKEKEDFGLKLYSNPKTNKLIERIDNHKLVKSHGRNQDRFMDFSMRFLAENNYFGKDAFFKCYKFYDTQEQDEFLQSNENNDDVKFHKGKVTTYIKYEEHLKNYSYWDCPFVEENNSMSVKISIGSEEKILKIQRNLMIYFLENALYNENVENQGYKLVNNYYRELKKDVEESIASLDLIKSNPDFKSKYKKILPKRLLHNYAPAKQDKAPENAFETLLKKADFREEQYKKLLKKAEHEKNKEDFVKRNKGKQFKLHFIRKACQMMYFKEKYNTLKEGNAAFEKKDPVIEKRKNKEHEFGHHKNLNITREEFNDYCKWMFAFNGNDSYKKYLRDLFSEKHFFDNQEYKNLFESSVNLEAFYAKTKELFKKWIETNKPTNNENRYTLENYKNLILQKQVFINVYHFSKYLIDKNLLNSENNVIQYKSLENVEYLISDFYFQSKLSIDQYKTCGKLFNKLKSNKLEDCLLYEIAYNYIDKKNVHKIDIQKILTSKIILTINDANTPYKISVPFNKLERYTEMIAIKNQNNLKARFLIDLPLYLSKNKIKKGKDSAGYEIIIKNDLEIEDINTINNKIINDSVKFTEVLMELEKYFILKDKCILSKNYIDNSEIPSLKQFSKVWIKENENEIINYRNIACHFHLPLLETFDNLLLNVEQKFIKEELQNVSTINDLSKPQEYLILLFIKFKHNNFYLNLFNKNESKTIKNDKEVKKNRVLQKFINQVILKKK (SEQ ID NO: 54) Myroides 17MKDILTTDTTEKQNRFYSHKIADKYFFGGYFNLASNNIYEVFEE odoratimimusVNKRNTFGKLAKRDNGNLKNYIIHVFKDELSISDFEKRVAIFASYFPILETVDKKSIKERNRTIDLTLSQRIRQFREMLISLVTAVDQLRNFYTHYHHSDIVIENKVLDFLNSSFVSTALHVKDKYLKTDKTKEFLKETIAAELDILIEAYKKKQIEKKNTRFKANKREDILNAIYNEAFWSFINDKDKDKDKETVVAKGADAYFEKNHHKSNDPDFALNISEKGIVYLLSFFLTNKEMDSLKANLTGFKGKVDRESGNSIKYMATQRIYSFHTYRGLKQKIRTSEEGVKETLLMQMIDELSKVPNVVYQHLSTTQQNSFIEDWNEYYKDYEDDVETDDLSRVTHPVIRKRYEDRFNYFAIRFLDEFFDFPTLRFQVHLGDYVHDRRTKQLGKVESDRIIKEKVTVFARLKDINSAKASYFHSLEEQDKEELDNKWTLFPNPSYDFPKEHTLQHQGEQKNAGKIGIYVKLRDTQYKEKAALEEARKSLNPKERSATKASKYDIITQIIEANDNVKSEKPLVFTGQPIAYLSMNDIHSMLFSLLTDNAELKKTPEEVEAKLIDQIGKQINEILSKDTDTKILKKYKDNDLKETDTDKITRDLARDKEEIEKLILEQKQRADDYNYTSSTKFNIDKSRKRKHLLFNAEKGKIGVWLANDIKRFMFKESKSKWKGYQHIELQKLFAYFDTSKSDLELILSNMVMVKDYPIELIDLVKKSRTLVDFLNKYLEARLEYIENVITRVKNSIGTPQFKTVRKECFTFLKKSNYTVVSLDKQVERILSMPLFIERGFMDDKPTMLEGKSYKQHKEKFADWFVHYKENSNYQNFYDTEVYEITTEDKREKAKVTKKIKQQQKNDVFTLMMVNYMLEEVLKLSSNDRLSLNELYQTKEERIVNKQVAKDTQERNKNYIWNKVVDLQLCDGLVHIDNVKLKDIGNFRKYENDSRVKEFLTYQSDIVWSAYLSNEVDSNKLYVIERQLDNYESIRSKELLKEVQEIECSVYNQVANKESLKQSGNENFKQYVLQGLLPIGMDVREMLILSTDVKFKKEEIIQLGQAGEVEQDLYSLIYIRNKFAHNQLPIKEFFDFCENNYRSISDNEYYAEYYMEIFRSIKEKYAN (SEQ ID NO: 55) Flavobacterium 18MSSKNESYNKQKTFNHYKQEDKYFFGGFLNNADDNLRQVGKE columnareFKTRINFNHNNNELASVFKDYFNKEKSVAKREHALNLLSNYFPVLERIQKHTNHNFEQTREIFELLLDTIKKLRDYYTHHYHKPITINPKIYDFLDDTLLDVLITIKKKKVKNDTSRELLKEKLRPELTQLKNQKREELIKKGKKLLEENLENAVFNHCLIPFLEENKTDDKQNKTVSLRKYRKSKPNEETSITLTQSGLVFLMSFFLHRKEFQVFTSGLERFKAKVNTIKEEEISLNKNNIVYMITHWSYSYYNFKGLKHRIKTDQGVSTLEQNNTTHSLTNTNTKEALLTQIVDYLSKVPNEIYETLSEKQQKEFEEDINEYMRENPENEDSTFSSIVSHKVIRKRYENKFNYFAMRFLDEYAELPTLRFMVNFGDYIKDRQKKILESIQFDSERIIKKEIHLFEKLSLVTEYKKNVYLKETSNIDLSRFPLFPNPSYVMANNNIPFYIDSRSNNLDEYLNQKKKAQSQNKKRNLTFEKYNKEQSKDAIIAMLQKEIGVKDLQQRSTIGLLSCNELPSMLYEVIVKDIKGAELENKIAQKIREQYQSIRDFTLDSPQKDNIPTTLIKTINTDSSVTFENQPIDIPRLKNALQKELTLTQEKLLNVKEHEIEVDNYNRNKNTYKFKNQPKNKVDDKKLQRKYVFYRNEIRQEANWLASDLIHFMKNKSLWKGYMHNELQSFLAFFEDKKNDCIALLETVFNLKEDCILTKGLKNLFLKHGNFIDFYKEYLKLKEDFLSTESTFLENGFIGLPPKILKKELSKRLKYIFIVFQKRQFIIKELEEKKNNLYADAINLSRGIFDEKPTMIPFKKPNPDEFASWFVASYQYNNYQSFYELTPDIVERDKKKKYKNLRAINKVKIQDYYLKLMVDTLYQDLFNQPLDKSLSDFYVSKAEREKIKADAKAYQKLNDSSLWNKVIHLSLQNNRITANPKLKDIGKYKRALQDEKIATLLTYDARTWTYALQKPEKENENDYKELHYTALNMELQEYEKVRSKELLKQVQELEKKILDKFYDFSNNASHPEDLEIEDKKGKRHPNFKLYITKALLKNESEIINLENIDIEILLKYYDYNTEELKEKIKNMDEDEKAKIINTKENYNKITNVLIKKALVLIIIRNKMAHNQYPPKFIYDLANRFVPKKEEEYFATYFNRVFETITKELWENKEKKDKTQV (SEQ ID NO: 56) Porphyromonas 20MTEQSERPYNGTYYTLEDKHFWAAFLNLARHNAYITLTHIDRQ sp.LAYSKADITNDQDVLSFKALWKNFDNDLERKSRLRSLILKHFSF COT-052LEGAAYGKKLFESKSSGNKSSKNKELTKKEKEELQANALSLDN OH4946LKSILFDFLQKLKDFRNYYSHYRHSESSELPLFDGNMLQRLYNVFDVSVQRVKRDHEHNDKVDPHRHFNHLVRKGKKDRYGHNDNPSFKHHFVDSEGMVTEAGLLFFVSLFLEKRDAIWMQKKIRGFKGGTETYQQMTNEVFCRSRISLPKLKLESLRTDDWMLLDMLNELVRCPKPLYDRLREDDRACFRVPVDILPDEDDTDGGGEDPFKNTLVRHQDRFPYFALRYFDLKKVFTSLRFHIDLGTYHFAIYKKMIGEQPEDRHLTRNLYGFGRIQDFAEEHRPEEWKRLVRDLDYFETGDKPYISQTTPHYHIEKGKIGLRFVPEGQHLWPSPEVGTTRTGRSKYAQDKRLTAEAFLSVHELMPMMFYYFLLREKYSEEVSAEKVQGRIKRVIEDVYAIYDAFARDEINTLKELDACLADKGIRRGHLPKQMIGILSQERKDMEEKVRKKLQEMIADTDHRLDMLDRQTDRKIRIGRKNAGLPKSGVIADWLVRDMMRFQPVAKDTSGKPLNNSKANSTEYRMLQRALALFGGEKERLTPYFRQMNLTGGNNPHPFLHETRWESHTNILSFYRSYLRARKAFLERIGRSDRVENCPFLLLKEPKTDRQTLVAGWKGEFHLPRGIFTEAVRDCLIEMGYDEVGSYREVGFMAKAVPLYFERACEDRVQPFYDSPFNVGNSLKPKKGRFLSKEDRAEEWERGKERFRDLEAWSHSAARRIKDAFAGIEYASPGNKKKIEQLLRDLSLWEAFESKLKVRADKINLAKLKKEILEAQEHPYHDFKSWQKFERELRLVKNQDIITWMMCRDLMEENKVEGLDTGTLYLKDIRPNVQEQGSLNVLNRVKPMRLPVVVYRADSRGHVHKEEAPLATVYIEERDTKLLKQGNFKSFVKDRRLNGLFSFVDTGGLAMEQYPISKLRVEYELAKYQTARVCVFELTLRLEESLLSRYPHLPDESFREMLESWSDPLLAKWPELHGKVRLLIAVRNAFSHNQYPMYDEAVESSIRKYDPSSPDAIEERMGLNIAHRLSEEVKQAKE TVERIIQA (SEQ ID NO: 57)Prevotella 21 MEDDKKTKESTNMLDNKHFWAAFLNLARHNVYITVNHINKVL intermediaELKNKKDQDIIIDNDQDILAIKTHWEKVNGDLNKTERLRELMTKHFPFLETAIYTKNKEDKEEVKQEKQAKAQSFDSLKHCLFLFLEKLQEARNYYSHYKYSESTKEPMLEKELLKKMYNIFDDNIQLVIKDYQHNKDINPDEDFKHLDRTEEEFNYYFTTNKKGNITASGLLFFVSLFLEKKDAIWMQQKLRGFKDNRESKKKMTHEVFCRSRMLLPKLRLESTQTQDWILLDMLNELIRCPKSLYERLQGEYRKKFNVPFDSADEDYDAEQEPFKNTLVRHQDRFPYFALRYFDYNEIFTNLRFQIDLGTYHFSIYKKLIGGQKEDRHLTHKLYGFERIQEFAKQNRTDEWKAIVKDFDTYETSEEPYISETAPHYHLENQKIGIRFRNDNDEIWPSLKINGENNEKRKYKLDKQYQAEAFLSVHELLPMMFYYLLLKKEEPNNDKKNASIVEGFIKREIRDIYKLYDAFANGEINNIDDLEKYCEDKGIPKRHLPKQMVAILYDEHKDMAEEAKRKQKEMVKDTKKLLATLEKQTQGEIEDGGRNIRLLKSGEIARWLVNDMMRFQPVQKDNEGNPLNNSKANSTEYQMLQRSLALYNKEEKPTRYFRQVNLINSSNPHPFLKWTKWEECNNILSFYRSYLTKKIEFLNKLKPEDWEKNQYFLKLKEPKTNRETLVQGWKNGFNLPRGIFTEPIREWFKRHQNDSEEYEKVETLDRVGLVTKVIPLFFKKEDSKDKEEYLKKDAQKEINNCVQPFYGFPYNVGNIHKPDEKDFLPSEERKKLWGDKKYKFKGYKAKVKSKKLTDKEKEEYRSYLEFQSWNKFERELRLVRNQDIVTWLLCTELIDKLKVEGLNVEELKKLRLKDIDTDTAKQEKNNILNRVMPMQLPVTVYEIDDSHNIVKDRPLHTVYIEETKTKLLKQGNFKALVKDRRLNGLFSFVDTSSETELKSNPISKSLVEYELGEYQNARIETIKDMLLLEETLIEKYKTLPTDNFSDMLNGWLEGKDEADKARFQNDVKLLVAVRNAFSHNQYPMRNRIAFANINPFSLSSADTSEEKKLDIANQLKDKTHKIIKRIIEIEKPIETK E (SEQ ID NO: 58)PIN17_0200 AFJ07523 MKMEDDKKTKESTNMLDNKHFWAAFLNLARHNVYITVNHIN [Prevotella KVLELKNKKDQDIIIDNDQDILAIKTHWEKVNGDLNKTERLREL intermediaMTKHFPFLETAIYTKNKEDKEEVKQEKQAKAQSFDSLKHCLFL 17]FLEKLQEARNYYSHYKYSESTKEPMLEKELLKKMYNIFDDNIQLVIKDYQHNKDINPDEDFKHLDRTEEEFNYYFTTNKKGNITASGLLFFVSLFLEKKDAIWMQQKLRGFKDNRESKKKMTHEVFCRSRMLLPKLRLESTQTQDWILLDMLNELIRCPKSLYERLQGEYRKKFNVPFDSADEDYDAEQEPFKNTLVRHQDRFPYFALRYFDYNEIFTNLRFQIDLGTYHFSIYKKLIGGQKEDRHLTHKLYGFERIQEFAKQNRTDEWKAIVKDFDTYETSEEPYISETAPHYHLENQKIGIRFRNDNDEIWPSLKTNGENNEKRKYKLDKQYQAEAFLSVHELLPMMFYYLLLKKEEPNNDKKNASIVEGFIKREIRDIYKLYDAFANGEINNIDDLEKYCEDKGIPKRHLPKQMVAILYDEHKDMAEEAKRKQKEMVKDTKKLLATLEKQTQGEIEDGGRNIRLLKSGEIARWLVNDMMRFQPVQKDNEGNPLNNSKANSTEYQMLQRSLALYNKEEKPTRYFRQVNLINSSNPHPFLKWTKWEECNNILSFYRSYLTKKIEFLNKLKPEDWEKNQYFLKLKEPKTNRETLVQGWKNGFNLPRGIFTEPIREWFKRHQNDSEEYEKVETLDRVGLVTKVIPLFFKKEDSKDKEEYLKKDAQKEINNCVQPFYGFPYNVGNIHKPDEKDFLPSEERKKLWGDKKYKFKGYKAKVKSKKLTDKEKEEYRSYLEFQSWNKFERELRLVRNQDIVTWLLCTELIDKLKVEGLNVEELKKLRLKDIDTDTAKQEKNNILNRVMPMQLPVTVYEIDDSHNIVKDRPLHTVYIEETKTKLLKQGNFKALVKDRRLNGLFSFVDTSSETELKSNPISKSLVEYELGEYQNARIETIKDMLLLEETLIEKYKTLPTDNFSDMLNGWLEGKDEADKARFQNDVKLLVAVRNAFSHNQYPMRNRIAFANINPFSLSSADTSEEKKLDIANQLKDKTHKIIKRIIEIEKPIETK E (SEQ ID NO: 59)Prevotella BAU18623 MEDDKKTTDSISYELKDKHFWAAFLNLARHNVYITVNHINKVLintermedia ELKNKKDQDIIIDNDQDILAIKTHWEKVNGDLNKTERLRELMTKHFPFLETAIYSKNKEDKEEVKQEKQAKAQSFDSLKHCLFLFLEKLQETRNYYSHYKYSESTKEPMLEKELLKKMYNIFDDNIQLVIKDYQHNKDINPDEDFKHLDRTEEDFNYYFTRNKKGNITESGLLFFVSLFLEKKDAIWMQQKLRGFKDNRESKKKMTHEVFCRSRMLLPKLRLESTQTQDWILLDMLNELIRCPKSLYERLQGEDREKFKVPFDPADEDYDAEQEPFKNTLVRHQDRFPYFALRYFDYNEIFTNLRFQIDLGTFHFSIYKKLIGGQKEDRHLTHKLYGFERIQEFAKQNRPDEWKAIVKDLDTYETSNERYISETTPHYHLENQKIGIRFRNDNDEIWPSLKTNGENNEKSKYKLDKQYQAEAFLSVHELLPMMFYYLLLKKEEPNNDKKNASIVEGFIKREIRDMYKLYDAFANGEINNIDDLEKYCEDKGIPKRHLPKQMVAILYDEHKDMVKEAKRKQRKMVKDTEKLLAALEKQTQEKTEDGGRNIRLLKSGEIARWLVNDMMRFQPVQKDNEGNPLNNSKANSTEYQMLQRSLALYNKEEKPTRYFRQVNLINSSNPHPFLKWTKWEECNNILSFYRSYLTKKIEFLNKLKPEDWEKNQYFLKLKEPKTNRETLVQGWKNGFNLPRGIFTEPIREWFKRHQNDSKEYEKVEALDRVGLVTKVIPLFFKKEDSKDKEEDLKKDAQKEINNCVQPFYSFPYNVGNIHKPDEKDFLHREERIELWDKKKDKFKGYKAKVKSKKLTDKEKEEYRSYLEFQSWNKFERELRLVRNQDIVTWLLCTELIDKLKVEGLNVEELKKLRLKDIDTDTAKQEKNNILNRVMPMQLPVTVYEIDDSHNIVKDRPLHTVYIEETKTKLLKQGNFKALVKDRRLNGLFSFVDTSSEAELKSNPISKSLVEYELGEYQNARIETIKDMLLLEETLIEKYKNLPTDNFSDMLNGWLEGKDEADKARFQNDVKLLVAVRNAFSHNQYPMRNRIAFANINPFSLSSADTSEEKKLDIANQLKDKTHKIIKRIIEIEKPIETKE (SEQ ID NO: 60) HMPREF6EFU31981 MQKQDKLFVDRKKNAIFAFPKYITIMENKEKPEPIYYELTDKHF 485_0083WAAFLNLARHNVYTTINHINRRLEIAELKDDGYMMGIKGSWNE [PrevotellaQAKKLDKKVRLRDLIMKHFPFLEAAAYEMTNSKSPNNKEQRE buccaeKEQSEALSLNNLKNVLFIFLEKLQVLRNYYSHYKYSEESPKPIFE ATCCTSLLKNMYKVFDANVRLVKRDYMHHENIDMQRDFTHLNRKK 33574]QVGRTKNIIDSPNFHYHFADKEGNMTIAGLLFFVSLFLDKKDAIWMQKKLKGFKDGRNLREQMTNEVFCRSRISLPKLKLENVQTKDWMQLDMLNELVRCPKSLYERLREKDRESFKVPFDIFSDDYNAEEEPFKNTLVRHQDRFPYFVLRYFDLNEIFEQLRFQIDLGTYHFSIYNKRIGDEDEVRHLTHHLYGFARIQDFAPQNQPEEWRKLVKDLDHFETSQEPYISKTAPHYHLENEKIGIKFCSAHNNLFPSLQTDKTCNGRSKFNLGTQFTAEAFLSVHELLPMMFYYLLLTKDYSRKESADKVEGIIRKEISNIYAIYDAFANNEINSIADLTRRLQNTNILQGHLPKQMISILKGRQKDMGKEAERKIGEMIDDTQRRLDLLCKQTNQKIRIGKRNAGLLKSGKIADWLVNDMMRFQPVQKDQNNIPINNSKANSTEYRMLQRALALFGSENFRLKAYFNQMNLVGNDNPHPFLAETQWEHQTNILSFYRNYLEARKKYLKGLKPQNWKQYQHFLILKVQKTNRNTLVTGWKNSFNLPRGIFTQPIREWFEKHNNSKRIYDQILSFDRVGFVAKAIPLYFAEEYKDNVQPFYDYPFNIGNRLKPKKRQFLDKKERVELWQKNKELFKNYPSEKKKTDLAYLDFLSWKKFERELRLIKNQDIVTWLMFKELFNMATVEGLKIGEIHLRDIDTNTANEESNNILNRIMPMKLPVKTYETDNKGNILKERPLATFYIEETETKVLKQGNFKALVKDRRLNGLFSFAETTDLNLEEHPISKLSVDLELIKYQTTRISIFEMTLGLEKKLIDKYSTLPTDSFRNMLERWLQCKANRPELKNYVNSLIAVRNAFSHNQYPMYDATLFAEVKKFTLFPSVDTKKIELNIAPQLLEIVGKAIKEIEKSENKN (SEQ ID NO: 61) HMPREF9 EGQ18444MKEEEKGKTPVVSTYNKDDKHFWAAFLNLARHNVYITVNHIN 144_1146KILGEGEINRDGYENTLEKSWNEIKDINKKDRLSKLIIKHFPFLE [PrevotellaVTTYQRNSADTTKQKEEKQAEAQSLESLKKSFFVFIYKLRDLRN pallensHYSHYKHSKSLERPKFEEDLQEKMYNIFDASIQLVKEDYKHNT ATCCDIKTEEDFKHLDRKGQFKYSFADNEGNITESGLLFFVSLFLEKK 700821]DAIWVQKKLEGFKCSNESYQKMTNEVFCRSRMLLPKLRLQSTQTQDWILLDMLNELIRCPKSLYERLREEDRKKFRVPIEIADEDYDAEQEPFKNALVRHQDRFPYFALRYFDYNEIFTNLRFQIDLGTYHFSIYKKQIGDYKESHHLTHKLYGFERIQEFTKQNRPDEWRKFVKTFNSFETSKEPYIPETTPHYHLENQKIGIRFRNDNDKIWPSLKTNSEKNEKSKYKLDKSFQAEAFLSVHELLPMMFYYLLLKTENTDNDNEIETKKKENKNDKQEKHKIEEIIENKITEIYALYDAFANGKINSIDKLEEYCKGKDIEIGHLPKQMIAILKSEHKDMATEAKRKQEEMLADVQKSLESLDNQINEEIENVERKNSSLKSGEIASWLVNDMMRFQPVQKDNEGNPLNNSKANSTEYQMLQRSLALYNKEEKPTRYFRQVNLIESSNPHPFLNNTEWEKCNNILSFYRSYLEAKKNFLESLKPEDWEKNQYFLMLKEPKTNCETLVQGWKNGFNLPRGIFTEPIRKWFMEHRKNITVAELKRVGLVAKVIPLFFSEEYKDSVQPFYNYLFNVGNINKPDEKNFLNCEERRELLRKKKDEFKKMTDKEKEENPSYLEFQSWNKFERELRLVRNQDIVTWLLCMELFNKKKIKELNVEKIYLKNINTNTTKKEKNTEEKNGEEKIIKEKNNILNRIMPMRLPIKVYGRENFSKNKKKKIRRNTFFTVYIEEKGTKLLKQGNFKALERDRRLGGLFSFVKTHSKAESKSNTISKSRVEYELGEYQKARIEIIKDMLALEETLIDKYNSLDTDNFHNMLTGWLKLKDEPDKASFQNDVDLLIAVRNAFSHNQYPMRNRIAFANINPFSLSSANTSEEKGLGIANQLKDKTHKTIEKIIEIEKPIETKE (SEQ ID NO: 62) HMPREF9 EHO08761MKDILTTDTTEKQNRFYSHKIADKYFFGGYFNLASNNIYEVFEE 714_02132VNKRNTFGKLAKRDNGNLKNYIIHVFKDELSISDFEKRVAIFAS [MyroidesYFPILETVDKKSIKERNRTIDLTLSQRIRQFREMLISLVTAVDQLR odoratimimusNFYTHYHHSEIVIENKVLDFLNSSLVSTALHVKDKYLKTDKTKE CCUGFLKETIAAELDILIEAYKKKQIEKKNTRFKANKREDILNAIYNEA 12901]FWSFINDKDKDKETVVAKGADAYFEKNHHKSNDPDFALNISEKGIVYLLSFFLTNKEMDSLKANLTGFKGKVDRESGNSIKYMATQRIYSFHTYRGLKQKIRTSEEGVKETLLMQMIDELSKVPNVVYQHLSTTQQNSFIEDWNEYYKDYEDDVETDDLSRVIHPVIRKRYEDRFNYFAIRFLDEFFDFPTLRFQVHLGDYVHDRRTKQLGKVESDRIIKEKVTVFARLKDINSAKANYFHSLEEQDKEELDNKWTLFPNPSYDFPKEHTLQHQGEQKNAGKIGIYVKLRDTQYKEKAALEEARKSLNPKERSATKASKYDIITQIIEANDNVKSEKPLVFTGQPIAYLSMNDIHSMLFSLLTDNAELKKTPEEVEAKLIDQIGKQINEILSKDTDTKILKKYKDNDLKETDTDKITRDLARDKEEIEKLILEQKQRADDYNYTSSTKFNIDKSRKRKHLLFNAEKGKIGVWLANDIKRFMTEEFKSKWKGYQHTELQKLFAYYDTSKSDLDLILSDMVMVKDYPIELIALVKKSRTLVDFLNKYLEARLGYMENVITRVKNSIGTPQFKTVRKECFTFLKKSNYTVVSLDKQVERILSMPLFIERGFMDDKPTMLEGKSYQQHKEKFADWFVHYKENSNYQNFYDTEVYEITTEDKREKAKVTKKIKQQQKNDVFTLMMVNYMLEEVLKLSSNDRLSLNELYQTKEERIVNKQVAKDTQERNKNYIWNKVVDLQLCEGLVRIDKVKLKDIGNFRKYENDSRVKEFLTYQSDIVWSAYLSNEVDSNKLYVIERQLDNYESIRSKELLKEVQEIECSVYNQVANKESLKQSGNENFKQYVLQGLVPIGMDVREMLILSTDVKFIKEEIIQLGQAGEVEQDLYSLIYIRNKFAHNQLPIKEFFDFCENNYRSISDNEYYAEYYMEIFRSIKEKYTS (SEQ ID NO: 63) HMPREF9 EKB06014MKDILTTDTTEKQNRFYSHKIADKYFFGGYFNLASNNIYEVFEE 711_00870VNKRNTFGKLAKRDNGNLKNYIIHVFKDELSISDFEKRVAIFAS [MyroidesYFPILETVDKKSIKERNRTIDLTLSQRIRQFREMLISLVTAVDQLR odoratimimusNFYTHYHHSEIVIENKVLDFLNSSLVSTALHVKDKYLKTDKTKE CCUGFLKETIAAELDILIEAYKKKQIEKKNTRFKANKREDILNAIYNEA 3837]FWSFINDKDKDKETVVAKGADAYFEKNHHKSNDPDFALNISEKGIVYLLSFFLTNKEMDSLKANLTGFKGKVDRESGNSIKYMATQRIYSFHTYRGLKQKIRTSEEGVKETLLMQMIDELSKVPNVVYQHLSTTQQNSFIEDWNEYYKDYEDDVETDDLSRVIHPVIRKRYEDRFNYFAIRFLDEFFDFPTLRFQVHLGDYVHDRRTKQLGKVESDRIIKEKVTVFARLKDINSAKASYFHSLEEQDKEELDNKWTLFPNPSYDFPKEHTLQHQGEQKNAGKIGIYVKLRDTQYKEKAALEEARKSLNPKERSATKASKYDIITQIIEANDNVKSEKPLVFTGQPIAYLSMNDIHSMLFSLLTDNAELKKTPEEVEAKLIDQIGKQINEILSKDTDTKILKKYKDNDLKETDTDKITRDLARDKEEIEKLILEQKQRADDYNYTSSTKFNIDKSRKRKHLLFNAEKGKIGVWLANDIKRFMFKESKSKWKGYQHTELQKLFAYFDTSKSDLELILSDMVMVKDYPIELIDLVRKSRTLVDFLNKYLEARLGYIENVITRVKNSIGTPQFKTVRKECFAFLKESNYTVASLDKQIERILSMPLFIERGFMDSKPTMLEGKSYQQHKEDFADWFVHYKENSNYQNFYDTEVYEIITEDKREQAKVTKKIKQQQKNDVFTLMMVNYMLEEVLKLPSNDRLSLNELYQTKEERIVNKQVAKDTQERNKNYIWNKVVDLQLCEGLVRIDKVKLKDIGNFRKYENDSRVKEFLTYQSDIVWSGYLSNEVDSNKLYVIERQLDNYESIRSKELLKEVQEIECIVYNQVANKESLKQSGNENFKQYVLQGLLPRGTDVREMLILSTDVKFKKEEIMQLGQVREVEQDLYSLIYIRNKFAHNQLPIKEFFDFCENNYRPISDNEYYAEYYMEIFRSIKEKYAS (SEQ ID NO: 64) HMPREF9 EKB54193MENKTSLGNNIYYNPFKPQDKSYFAGYFNAAMENTDSVFRELG 699_02005KRLKGKEYTSENFFDAIFKENISLVEYERYVKLLSDYFPMARLL [BergeyellaDKKEVPIKERKENFKKNFKGIIKAVRDLRNFYTHKEHGEVEITD zoohelcumEIFGVLDEMLKSTVLTVKKKKVKTDKTKEILKKSIEKQLDILCQ ATCCKKLEYLRDTARKIEEKRRNQRERGEKELVAPFKYSDKRDDLIA 43767]AIYNDAFDVYIDKKKDSLKESSKAKYNTKSDPQQEEGDLKIPISKNGVVFLLSLFLTKQEIHAFKSKIAGFKATVIDEATVSEATVSHGKNSICFMATHEIFSHLAYKKLKRKVRTAEINYGEAENAEQLSVYAKETLMMQMLDELSKVPDVVYQNLSEDVQKTFIEDWNEYLKENNGDVGTMEEEQVIHPVIRKRYEDKFNYFAIRFLDEFAQFPTLRFQVHLGNYLHDSRPKENLISDRRIKEKITVFGRLSELEHKKALFIKNTETNEDREHYWEIFPNPNYDFPKENISVNDKDFPIAGSILDREKQPVAGKIGIKVKLLNQQYVSEVDKAVKAHQLKQRKASKPSIQNIIEEIVPINESNPKEAIVFGGQPTAYLSMNDIHSILYEFFDKWEKKKEKLEKKGEKELRKEIGKELEKKIVGKIQAQIQQIIDKDTNAKILKPYQDGNSTAIDKEKLIKDLKQEQNILQKLKDEQTVREKEYNDFIAYQDKNREINKVRDRNHKQYLKDNLKRKYPEAPARKEVLYYREKGKVAVWLANDIKRFMPTDFKNEWKGEQHSLLQKSLAYYEQCKEELKNLLPEKVFQHLPFKLGGYFQQKYLYQFYTCYLDKRLEYISGLVQQAENFKSENKVFKKVENECFKFLKKQNYTHKELDARVQSILGYPIFLERGFMDEKPTIIKGKTFKGNEALFADWFRYYKEYQNFQTFYDTENYPLVELEKKQADRKRKTKIYQQKKNDVFTLLMAKHIFKSVFKQDSIDQFSLEDLYQSREERLGNQERARQTGERNTNYIWNKTVDLKLCDGKITVENVKLKNVGDFIKYEYDQRVQAFLKYEENIEWQAFLIKESKEEENYPYVVEREIEQYEKVRREELLKEVHLIEEYILEKVKDKEILKKGDNQNFKYYILNGLLKQLKNEDVESYKVFNLNTEPEDVNINQLKQEATDLEQKAFVLTYIRNKFAHNQLPKKEFWDYCQEKYGKIEKEKTYAEYFAEVFKKEKE ALIK (SEQ ID NO: 65) HMPREF9EKY00089 MMEKENVQGSHIYYEPTDKCFWAAFYNLARHNAYLTIAHINSF 151_01387VNSKKGINNDDKVLDIIDDWSKFDNDLLMGARLNKLILKHFPFL [PrevotellaKAPLYQLAKRKTRKQQGKEQQDYEKKGDEDPEVIQEAIANAFK saccharolyticaMANVRKTLHAFLKQLEDLRNHFSHYNYNSPAKKMEVKFDDGF F0055]CNKLYYVFDAALQMVKDDNRMNPEINMQTDFEHLVRLGRNRKIPNTFKYNFTNSDGTINNNGLLFFVSLFLEKRDAIWMQKKIKGFKGGTENYMRMTNEVFCRNRMVIPKLRLETDYDNHQLMFDMLNELVRCPLSLYKRLKQEDQDKFRVPIEFLDEDNEADNPYQENANSDENPTEETDPLKNTLVRHQHRFPYFVLRYFDLNEVFKQLRFQINLGCYHFSIYDKTIGERTEKRHLTRTLFGFDRLQNFSVKLQPEHWKNMVKHLDTEESSDKPYLSDAMPHYQIENEKIGIHFLKTDTEKKETVWPSLEVEEVSSNRNKYKSEKNLTADAFLSTHELLPMMFYYQLLSSEEKTRAAAGDKVQGVLQSYRKKIFDIYDDFANGTINSMQKLDERLAKDNLLRGNMPQQMLAILEHQEPDMEQKAKEKLDRLITETKKRIGKLEDQFKQKVRIGKRRADLPKVGSIADWLVNDMMRFQPAKRNADNTGVPDSKANSTEYRLLQEALAFYSAYKDRLEPYFRQVNLIGGTNPHPFLHRVDWKKCNHLLSFYHDYLEAKEQYLSHLSPADWQKHQHFLLLKVRKDIQNEKKDWKKSLVAGWKNGFNLPRGLFTESIKTWFSTDADKVQITDTKLFENRVGLIAKLIPLYYDKVYNDKPQPFYQYPFNINDRYKPEDTRKRFTAASSKLWNEKKMLYKNAQPDSSDKIEYPQYLDFLSWKKLERELRMLRNQDMMVWLMCKDLFAQCTVEGVEFADLKLSQLEVDVNVQDNLNVLNNVSSMILPLSVYPSDAQGNVLRNSKPLHTVYVQENNTKLLKQGNFKSLLKDRRLNGLFSFIAAEGEDLQQHPLTKNRLEYELSIYQTMRISVFEQTLQLEKAILTRNKTLCGNNFNNLLNSWSEHRTDKKTLQPDIDFLIAVRNAFSHNQYPMSTNTVMQGIEKFNIQTPKLEEKDGLGIASQLAKKTKDAASRLQNIINGGTN (SEQ ID NO: 66) A343_1752 EOA10535MTEQNEKPYNGTYYTLEDKHFWAAFFNLARHNAYITLTHIDRQ [PorphyromonasLAYSKADITNDEDILFFKGQWKNLDNDLERKARLRSLILKHFSF gingivalisLEGAAYGKKLFESQSSGNKSSKKKELTKKEKEELQANALSLDN JCVILKSILFDFLQKLKDFRNYYSHYRHPESSELPLFDGNMLQRLYNV SC001]FDVSVQRVKRDHEHNDKVDPHRHFNHLVRKGKKDRCGNNDNPFFKHHFVDREEKVTEAGLLFFVSLFLEKRDAIWMQKKIRGFKGGTETYQQMTNEVFCRSRISLPKLKLESLRTDDWMLLDMLNELVRCPKSLYDRLREEDRARFRVPVDILSDEDDTDGTEEDPFKNTLVRHQDRFPYFALRYFDLKKVFTSLRFHIDLGTYHFAIYKKNIGEQPEDRHLTRNLYGFGRIQDFAEEHRPEEWKRLVRDLDYFETGDKPYITQTTPHYHIEKGKIGLRFVPEGQLLWPSPEVGATRTGRSKYAQDKRFTAEAFLSVHELMPMMFYYFLLREKYSEEASAERVQGRIKRVIEDVYAVYDAFARGEIDTLDRLDACLADKGIRRGHLPRQMIAILSQEHKDMEEKVRKKLQEMIADTDHRLDMLDRQTDRKIRIGRKNAGLPKSGVIADWLVRDMMRFQPVAKDTSGKPLNNSKANSTEYRMLQRALALFGGEKERLTPYFRQMNLTGGNNPHPFLHETRWESHTNILSFYRSYLKARKAFLQSIGRSDRVENHRFLLLKEPKTDRQTLVAGWKGEFHLPRGIFTEAVRDCLIEMGLDEVGSYKEVGFMAKAVPLYFERACKDRVQPFYDYPFNVGNSLKPKKGRFLSKEKRAEEWESGKERFRDLEAWSHSAARRIEDAFAGIENASRENKKKIEQLLQDLSLWETFESKLKVKADKINIAKLKKEILEAKEHPYLDFKSWQKFERELRLVKNQDIITWMMCRDLMEENKVEGLDTGTLYLKDIRTDVHEQGSLNVLNRVKPMRLPVVVYRADSRGHVHKEQAPLATVYIEERDTKLLKQGNFKSFVKDRRLNGLFSFVDTGALAMEQYPISKLRVEYELAKYQTARVCAFEQTLELEESLLTRYPHLPDKNFRKMLESWSDPLLDKWPDLHGNVRLLIAVRNAFSHNQYPMYDETLFSSIRKYDPSSPDAIEERMGLNIAHRLSEEVKQAK EMVERIIQA (SEQ ID NO: 67)HMPREF1 ERI81700 MESIKNSQKSTGKTLQKDPPYFGLYLNMALLNVRKVENHIRKW 981_03090LGDVALLPEKSGFHSLLTTDNLSSAKWTRFYYKSRKFLPFLEMF [BacteroidesDSDKKSYENRRETTECLDTIDRQKISSLLKEVYGKLQDIRNAFS pyogenesHYHIDDQSVKHTALIISSEMHRFIENAYSFALQKTRARFTGVFVE F0041]TDFLQAEEKGDNKKFFAIGGNEGIKLKDNALIFLICLFLDREEAFKFLSRATGFKSTKEKGFLAVRETFCALCCRQPHERLLSVNPREALLMDMLNELNRCPDILFEMLDEKDQKSFLPLLGEEEQAHILENSLNDELCEAIDDPFEMIASLSKRVRYKNRFPYLMLRYIEEKNLLPFIRFRIDLGCLELASYPKKMGEENNYERSVTDHAMAFGRLTDFHNEDAVLQQITKGITDEVRFSLYAPRYAIYNNKIGFVRTGGSDKISFPTLKKKGGEGHCVAYTLQNTKSFGFISIYDLRKILLLSFLDKDKAKNIVSGLLEQCEKHWKDLSENLFDAIRTELQKEFPVPLIRYTLPRSKGGKLVSSKLADKQEKYESEFERRKEKLTEILSEKDFDLSQIPRRMIDEWLNVLPTSREKKLKGYVETLKLDCRERLRVFEKREKGEHPVPPRIGEMATDLAKDIIRMVIDQGVKQRITSAYYSEIQRCLAQYAGDDNRRHLDSIIRELRLKDTKNGHPFLGKVLRPGLGHTEKLYQRYFEEKKEWLEATFYPAASPKRVPRFVNPPTGKQKELPLIIRNLMKERPEWRDWKQRKNSHPIDLPSQLFENEICRLLKDKIGKEPSGKLKWNEMFKLYWDKEFPNGMQRFYRCKRRVEVFDKVVEYEYSEEGGNYKKYYEALIDEVVRQKISSSKEKSKLQVEDLTLSVRRVFKRAINEKEYQLRLLCEDDRLLFMAVRDLYDWKEAQLDLDKIDNMLGEPVSVSQVIQLEGGQPDAVIKAECKLKDVSKLMRYCYDGRVKGLMPYFANHEATQEQVEMELRHYEDHRRRVFNWVFALEKSVLKNEKLRRFYEESQGGCEHRRCIDALRKASLVSEEEYEFLVHIRNKSAHNQFPDLEIGKLPPNVTSGFCECIWSKYKAIICRIIPFIDPERRFFGKLLEQK (SEQ ID NO: 68) HMPREF1 ERJ65637MNTVPASENKGQSRTVEDDPQYFGLYLNLARENLIEVESHVRIK 553_02065FGKKKLNEESLKQSLLCDHLLSVDRWTKVYGHSRRYLPFLHYF [PorphyromonasDPDSQIEKDHDSKTGVDPDSAQRLIRELYSLLDFLRNDFSHNRL gingivalisDGTTFEHLEVSPDISSFITGTYSLACGRAQSRFADFFKPDDFVLA F0568]KNRKEQLISVADGKECLTVSGLAFFICLFLDREQASGMLSRIRGFKRTDENWARAVHETFCDLCIRHPHDRLESSNTKEALLLDMLNELNRCPRILYDMLPEEERAQFLPALDENSMNNLSENSLNEESRLLWDGSSDWAEALTKRIRHQDRFPYLMLRFIEEMDLLKGIRFRVDLGEIELDSYSKKVGRNGEYDRTITDHALAFGKLSDFQNEEEVSRMISGEASYPVRFSLFAPRYAIYDNKIGYCHTSDPVYPKSKTGEKRALSNPRSMGFISVHDLRKLLLMELLCEGSFSRMQSDFLRKANRILDETAEGKLQFSALFPEMRHRFIPPQNPKSKDRREKAETTLEKYKQEIKGRKDKLNSQLLSAFDMDQRQLPSRLLDEWMNIRPASHSVKLRTYVKQLNEDCRLRLQKFRKDGDGKARAIPLVGEMATFLSQDIVRMIISEETKKLITSAYYNEMQRSLAQYAGEENRHQFRAIVAELRLLDPSSGHPFLSATMETAHRYTEDFYKCYLEKKREWLAKTFYRPEQDENTKRRISVFFVPDGEARKLLPLLIRRRMKEQNDLQDWIRNKQAHPIDLPSHLFDSKIMELLKVKDGKKKWNEAFKDWWSTKYPDGMQPFYGLRRELNIHGKSVSYIPSDGKKFADCYTHLMEKTVQDKKRELRTAGKPVPPDLAADIKRSFHRAVNEREFMLRLVQEDDRLMLMAINKMMTDREEDILPGLKNIDSILDEENQFSLAVHAKVLEKEGEGGDNSLSLVPATIEIKSKRKDWSKYIRYRYDRRVPGLMSHFPEHKATLDEVKTLLGEYDRCRIKIFDWAFALEGAIMSDRDLKPYLHESSSREGKSGEHSTLVKMLVEKKGCLTPDESQYLILIRNKAAHNQFPCAAEMPLIYRDVSAKVGSIEGSSAKDLPEGSSLVDSLWKKYEMIIRKILPILDPENRFFGKLLNNMSQPINDL (SEQ ID NO: 69) HMPREF1ERJ81987 MNTVPASENKGQSRTVEDDPQYFGLYLNLARENLIEVESHVRIK 988_01768FGKKKLNEESLKQSLLCDHLLSVDRWTKVYGHSRRYLPFLHYF [PorphyromonasDPDSQIEKDHDSKTGVDPDSAQRLIRELYSLLDFLRNDFSHNRL gingivalisDGTTFEHLEVSPDISSFITGTYSLACGRAQSRFADFFKPDDFVLA F0185]KNRKEQLISVADGKECLTVSGLAFFICLFLDREQASGMLSRIRGFKRTDENWARAVHETFCDLCIRHPHDRLESSNTKEALLLDMLNELNRCPRILYDMLPEEERAQFLPALDENSMNNLSENSLNEESRLLWDGSSDWAEALTKRIRHQDRFPYLMLRFIEEMDLLKGIRFRVDLGEIELDSYSKKVGRNGEYDRTITDHALAFGKLSDFQNEEEVSRMISGEASYPVRFSLFAPRYAIYDNKIGYCHTSDPVYPKSKTGEKRALSNPQSMGFISVHDLRKLLLMELLCEGSFSRMQSGFLRKANRILDETAEGKLQFSALFPEMRHRFIPPQNPKSKDRREKAETTLEKYKQEIKGRKDKLNSQLLSAFDMNQRQLPSRLLDEWMNIRPASHSVKLRTYVKQLNEDCRLRLRKFRKDGDGKARAIPLVGEMATFLSQDIVRMIISEETKKLITSAYYNEMQRSLAQYAGEENRRQFRAIVAELHLLDPSSGHPFLSATMETAHRYTEDFYKCYLEKKREWLAKTFYRPEQDENTKRRISVFFVPDGEARKLLPLLIRRRMKEQNDLQDWIRNKQAHPIDLPSHLFDSKIMELLKVKDGKKKWNEAFKDWWSTKYPDGMQPFYGLRRELNIHGKSVSYIPSDGKKFADCYTHLMEKTVQDKKRELRTAGKPVPPDLAADIKRSFHRAVNEREFMLRLVQEDDRLMLMAINKMMTDREEDILPGLKNIDSILDEENQFSLAVHAKVLEKEGEGGDNSLSLVPATIEIKSKRKDWSKYIRYRYDRRVPGLMSHFPEHKATLDEVKTLLGEYDRCRIKIFDWAFALEGAIMSDRDLKPYLHESSSREGKSGEHSTLVKMLVEKKGCLTPDESQYLILIRNKAAHNQFPCAAEMPLIYRDVSAKVGSIEGSSAKDLPEGSSLVDSLWKKYEMIIRKILPILDHENRFFGKLLNNMSQPINDL (SEQ ID NO: 70) HMPREF1ERJ87335 MNTVPASENKGQSRTVEDDPQYFGLYLNLARENLIEVESHVRIK 990_01800FGKKKLNEESLKQSLLCDHLLSVDRWTKVYGHSRRYLPFLHYF [PorphyromonasDPDSQIEKDHDSKTGVDPDSAQRLIRELYSLLDFLRNDFSHNRL gingivalisDGTTFEHLEVSPDISSFITGTYSLACGRAQSRFADFFKPDDFVLA W4087]KNRKEQLISVADGKECLTVSGLAFFICLFLDREQASGMLSRIRGFKRTDENWARAVHETFCDLCIRHPHDRLESSNTKEALLLDMLNELNRCPRILYDMLPEEERAQFLPALDENSMNNLSENSLNEESRLLWDGSSDWAEALTKRIRHQDRFPYLMLRFIEEMDLLKGIRFRVDLGEIELDSYSKKVGRNGEYDRTITDHALAFGKLSDFQNEEEVSRMISGEASYPVRFSLFAPRYAIYDNKIGYCHTSDPVYPKSKTGEKRALSNPRSMGFISVHDLRKLLLMELLCEGSFSRMQSDFLRKANRILDETAEGKLQFSALFPEMRHRFIPPQNPKSKDRREKAETTLEKYKQEIKGRKDKLNSQLLSAFDMDQRQLPSRLLDEWMNIRPASHSVKLRTYVKQLNEDCRLRLQKFRKDGDGKARAIPLVGEMATFLSQDIVRMIISEETKKLITSAYYNEMQRSLAQYAGEENRHQFRAIVAELRLLDPSSGHPFLSATMETAHRYTEDFYKCYLEKKREWLAKTFYRPEQDENTKRRISVFFVPDGEARKLLPLLIRRRMKEQNDLQDWIRNKQAHPIDLPSHLFDSKVMELLKVKDGKKKWNEAFKDWWSTKYPDGMQPFYGLRRELNIHGKSVSYIPSDGKKFADCYTHLMEKTVRDKKRELRTAGKPVPPDLAAYIKRSFHRAVNEREFMLRLVQEDDRLMLMAINKIMTDREEDILPGLKNIDSILDKENQFSLAVHAKVLEKEGEGGDNSLSLVPATIEIKSKRKDWSKYIRYRYDRRVPGLMSHFPEHKATLDEVKTLLGEYDRCRIKIFDWAFALEGAIMSDRDLKPYLHESSSREGKSGEHSTLVKMLVEKKGCLTPDESQYLILIRNKAAHNQFPCAAEIPLIYRDVSAKVGSIEGSSAKDLPEGSSLVDSLWKKYEMIIRKILPILDPENRFFGKLLNNMSQPINDL (SEQ ID NO: 71) M573_117042KJJ86756 MKMEDDKKTTESTNMLDNKHFWAAFLNLARHNVYITVNHINK [PrevotellaVLELKNKKDQDIIIDNDQDILAIKTHWEKVNGDLNKTERLRELM intermediaTKHFPFLETAIYTKNKEDKEEVKQEKQAEAQSLESLKDCLFLFL ZT]EKLQEARNYYSHYKYSESTKEPMLEEGLLEKMYNIFDDNIQLVIKDYQHNKDINPDEDFKHLDRKGQFKYSFADNEGNITESGLLFFVSLFLEKKDAIWMQQKLTGFKDNRESKKKMTHEVFCRRRMLLPKLRLESTQTQDWILLDMLNELIRCPKSLYERLQGEYRKKFNVPFDSADEDYDAEQEPFKNTLVRHQDRFPYFALRYFDYNEIFTNLRFQIDLGTYHFSIYKKLIGGQKEDRHLTHKLYGFERIQEFAKQNRPDEWKALVKDLDTYETSNERYISETTPHYHLENQKIGIRFRNGNKEIWPSLKTNGENNEKSKYKLDKPYQAEAFLSVHELLPMMFYYLLLKKEEPNNDKKNASIVEGFIKREIRDMYKLYDAFANGEINNIGDLEKYCEDKGIPKRHLPKQMVAILYDEPKDMVKEAKRKQKEMVKDTKKLLATLEKQTQEEIEDGGRNIRLLKSGEIARWLVNDMMRFQPVQKDNEGNPLNNSKANSTEYQMLQRSLALYNKEEKPTRYFRQVNLINSSNPHPFLKWTKWEECNNILSFYRNYLTKKIEFLNKLKPEDWEKNQYFLKLKEPKTNRETLVQGWKNGFNLPRGIFTEPIREWFKRHQNDSKEYEKVEALKRVGLVTKVIPLFFKEEYFKEDAQKEINNCVQPFYSFPYNVGNIHKPDEKDFLPSEERKKLWGDKKDKFKGYKAKVKSKKLTDKEKEEYRSYLEFQSWNKFERELRLVRNQDIVTWLLCTELIDKMKVEGLNVEELQKLRLKDIDTDTAKQEKNNILNRIMPMQLPVTVYEIDDSHNIVKDRPLHTVYIEETKTKLLKQGNFKALVKDRRLNGLFSFVDTSSKAELKDKPISKSVVEYELGEYQNARIETIKDMLLLEKTLIKKYEKLPTDNFSDMLNGWLEGKDESDKARFQNDVKLLVAVRNAFSHNQYPMRNRIAFANINPFSLSSADISEEKKLDIANQLKDKTHKIIKKIIEIEKPIETKE (SEQ ID NO: 72) Prevotella WP_MQKQDKLFVDRKKNAIFAFPKYITIMENQEKPEPIYYELTDKHF buccae 004343581WAAFLNLARHNVYTTINHINRRLEIAELKDDGYMMDIKGSWNEQAKKLDKKVRLRDLIMKHFPFLEAAAYEITNSKSPNNKEQREKEQSEALSLNNLKNVLFIFLEKLQVLRNYYSHYKYSEESPKPIFETSLLKNMYKVFDANVRLVKRDYMHHENIDMQRDFTHLNRKKQVGRTKNIIDSPNFHYHFADKEGNMTIAGLLFFVSLFLDKKDAIWMQKKLKGFKDGRNLREQMTNEVFCRSRISLPKLKLENVQTKDWMQLDMLNELVRCPKSLYERLREKDRESFKVPFDIFSDDYDAEEEPFKNTLVRHQDRFPYFVLRYFDLNEIFEQLRFQIDLGTYHFSIYNKRIGDEDEVRHLTHHLYGFARIQDFAQQNQPEVWRKLVKDLDYFEASQEPYIPKTAPHYHLENEKIGIKFCSTHNNLFPSLKTEKTCNGRSKFNLGTQFTAEAFLSVHELLPMMFYYLLLTKDYSRKESADKVEGIIRKEISNIYAIYDAFANGEINSIADLTCRLQKTNILQGHLPKQMISILEGRQKDMEKEAERKIGEMIDDTQRRLDLLCKQTNQKIRIGKRNAGLLKSGKIADWLVNDMMRFQPVQKDQNNIPINNSKANSTEYRMLQRALALFGSENFRLKAYFNQMNLVGNDNPHPFLAETQWEHQTNILSFYRNYLEARKKYLKGLKPQNWKQYQHFLILKVQKTNRNTLVTGWKNSFNLPRGIFTQPIREWFEKHNNSKRIYDQILSFDRVGFVAKAIPLYFAEEYKDNVQPFYDYPFNIGNKLKPQKGQFLDKKERVELWQKNKELFKNYPSEKKKTDLAYLDFLSWKKFERELRLIKNQDIVTWLMFKELFNMATVEGLKIGEIHLRDIDTNTANEESNNILNRIMPMKLPVKTYETDNKGNILKERPLATFYIEETETKVLKQGNFKVLAKDRRLNGLLSFAETTDIDLEKNPITKLSVDHELIKYQTTRISIFEMTLGLEKKLINKYPTLPTDSFRNMLERWLQCKANRPELKNYVNSLIAVRNAFSHNQYPMYDATLFAEVKKFTLFPSVDTKKIELNIAPQLLEIVGKAIKEIEKSENKN (SEQ ID NO: 73) PorphyromonasWP_ MNTVPASENKGQSRTVEDDPQYFGLYLNLARENLIEVESHVRIK gingivalis 005873511FGKKKLNEESLKQSLLCDHLLSVDRWTKVYGHSRRYLPFLHYFDPDSQIEKDHDSKTGVDPDSAQRLIRELYSLLDFLRNDFSHNRLDGTTFEHLEVSPDISSFITGTYSLACGRAQSRFADFFKPDDFVLAKNRKEQLISVADGKECLTVSGLAFFICLFLDREQASGMLSRIRGFKRTDENWARAVHETFCDLCIRHPHDRLESSNTKEALLLDMLNELNRCPRILYDMLPEEERAQFLPALDENSMNNLSENSLNEESRLLWDGSSDWAEALTKRIRHQDRFPYLMLRFIEEMDLLKGIRFRVDLGEIELDSYSKKVGRNGEYDRTITDHALAFGKLSDFQNEEEVSRMISGEASYPVRFSLFAPRYAIYDNKIGYCHTSDPVYPKSKTGEKRALSNPQSMGFISVHNLRKLLLMELLCEGSFSRMQSDFLRKANRILDETAEGKLQFSALFPEMRHRFIPPQNPKSKDRREKAETTLEKYKQEIKGRKDKLNSQLLSAFDMNQRQLPSRLLDEWMNIRPASHSVKLRTYVKQLNEDCRLRLRKFRKDGDGKARAIPLVGEMATFLSQDIVRMIISEETKKLITSAYYNEMQRSLAQYAGEENRRQFRAIVAELHLLDPSSGHPFLSATMETAHRYTEDFYKCYLEKKREWLAKTFYRPEQDENTKRRISVFFVPDGEARKLLPLLIRRRMKEQNDLQDWIRNKQAHPIDLPSHLFDSKIMELLKVKDGKKKWNEAFKDWWSTKYPDGMQPFYGLRRELNIHGKSVSYIPSDGKKFADCYTHLMEKTVQDKKRELRTAGKPVPPDLAADIKRSFHRAVNEREFMLRLVQEDDRLMLMAINKMMTDREEDILPGLKNIDSILDEENQFSLAVHAKVLEKEGEGGDNSLSLVPATIEIKSKRKDWSKYIRYRYDRRVPGLMSHFPEHKATLDEVKTLLGEYDRCRIKIFDWAFALEGAIMSDRDLKPYLHESSSREGKSGEHSTLVKMLVEKKGCLTPDESQYLILIRNKAAHNQFPCAAEMPLIYRDVSAKVGSIEGSSAKDLPEGSSLVDSLWKKYEMIIRKILPILDPENRFFGKLLNNMSQPINDL (SEQ ID NO: 74)Porphyromonas WP_ MTEQNEKPYNGTYYTLEDKHFWAAFFNLARHNAYITLAHIDRQ gingivalis500874195 LAYSKADITNDEDILFFKGQWKNLDNDLERKARLRSLILKHFSFLEGAAYGKKLFESQSSGNKSSKKKELTKKEKEELQANALSLDNLKSILFDFLQKLKDFRNYYSHYRHPESSELPLFDGNMLQRLYNVFDVSVQRVKRDHEHNDKVDPHRHFNHLVRKGKKDKYGNNDNPFFKHHFVDREEKVTEAGLLFFVSLFLEKRDAIWMQKKIRGFKGGTEAYQQMTNEVFCRSRISLPKLKLESLRTDDWMLLDMLNELVRCPKSLYDRLREEDRARFRVPVDILSDEDDTDGTEEDPFKNTLVRHQDRFPYFALRYFDLKKVFTSLRFHIDLGTYHFAIYKKNIGEQPEDRHLTRNLYGFGRIQDFAEEHRPEEWKRLVRDLDYFETGDKPYITQTTPHYHIEKGKIGLRFVPEGQLLWPSPEVGATRTGRSKYAQDKRFTAEAFLSVHELMPMMFYYFLLREKYSEEASAEKVQGRIKRVIEDVYAVYDAFARDEINTRDELDACLADKGIRRGHLPRQMIAILSQEHKDMEEKVRKKLQEMIADTDHRLDMLDRQTDRKIRIGRKNAGLPKSGVIADWLVRDMMRFQPVAKDTSGKPLNNSKANSTEYRMLQRALALFGGEKERLTPYFRQMNLTGGNNPHPFLHETRWESHTNILSFYRSYLKARKAFLQSIGRSDREENHRFLLLKEPKTDRQTLVAGWKSEFHLPRGIFTEAVRDCLIEMGYDEVGSYKEVGFMAKAVPLYFERACKDRVQPFYDYPFNVGNSLKPKKGRFLSKEKRAEEWESGKERFRDLEAWSHSAARRIEDAFVGIEYASWENKKKIEQLLQDLSLWETFESKLKVKADKINIAKLKKEILEAKEHPYHDFKSWQKFERELRLVKNQDIITWMMCRDLMEENKVEGLDTGTLYLKDIRTDVQEQGSLNVLNHVKPMRLPVVVYRADSRGHVHKEEAPLATVYIEERDTKLLKQGNFKSFVKDRRLNGLFSFVDTGALAMEQYPISKLRVEYELAKYQTARVCAFEQTLELEESLLTRYPHLPDESFREMLESWSDPLLDKWPDLQREVRLLIAVRNAFSHNQYPMYDETIFSSIRKYDPSSLDAIEERMGLNIAHRLSEEVKLAKEMV ERIIQA (SEQ ID NO: 75)Prevotella WP_ MKEEEKGKTPVVSTYNKDDKHFWAAFLNLARHNVYITVNHIN pallens006044833 KILGEGEINRDGYENTLEKSWNEIKDINKKDRLSKLIIKHFPFLEVTTYQRNSADTTKQKEEKQAEAQSLESLKKSFFVFIYKLRDLRNHYSHYKHSKSLERPKFEEDLQEKMYNIFDASIQLVKEDYKHNTDIKTEEDFKHLDRKGQFKYSFADNEGNITESGLLFFVSLFLEKKDAIWVQKKLEGFKCSNESYQKMTNEVFCRSRMLLPKLRLQSTQTQDWILLDMLNELIRCPKSLYERLREEDRKKFRVPIEIADEDYDAEQEPFKNALVRHQDRFPYFALRYFDYNEIFTNLRFQIDLGTYHFSIYKKQIGDYKESHHLTHKLYGFERIQEFTKQNRPDEWRKFVKTFNSFETSKEPYIPETTPHYHLENQKIGIRFRNDNDKIWPSLKTNSEKNEKSKYKLDKSFQAEAFLSVHELLPMMFYYLLLKTENTDNDNEIETKKKENKNDKQEKHKIEEIIENKITEIYALYDAFANGKINSIDKLEEYCKGKDIEIGHLPKQMIAILKSEHKDMATEAKRKQEEMLADVQKSLESLDNQINEEIENVERKNSSLKSGEIASWLVNDMMRFQPVQKDNEGNPLNNSKANSTEYQMLQRSLALYNKEEKPTRYFRQVNLIESSNPHPFLNNTEWEKCNNILSFYRSYLEAKKNFLESLKPEDWEKNQYFLMLKEPKTNCETLVQGWKNGFNLPRGIFTEPIRKWFMEHRKNITVAELKRVGLVAKVIPLFFSEEYKDSVQPFYNYLFNVGNINKPDEKNFLNCEERRELLRKKKDEFKKMTDKEKEENPSYLEFQSWNKFERELRLVRNQDIVTWLLCMELFNKKKIKELNVEKIYLKNINTNTTKKEKNTEEKNGEEKIIKEKNNILNRIMPMRLPIKVYGRENFSKNKKKKIRRNTFFTVYIEEKGTKLLKQGNFKALERDRRLGGLFSFVKTHSKAESKSNTISKSRVEYELGEYQKARIEIIKDMLALEETLIDKYNSLDTDNFHNMLTGWLKLKDEPDKASFQNDVDLLIAVRNAFSHNQYPMRNRIAFANINPFSLSSANTSEEKGLGIANQLKDKTHKTIEKIIEIEKPIETKE (SEQ ID NO: 76) Myroides WP_MKDILTTDTTEKQNRFYSHKIADKYFFGGYFNLASNNIYEVFEE odoratimimus 006261414VNKRNTFGKLAKRDNGNLKNYIIHVFKDELSISDFEKRVAIFASYFPILETVDKKSIKERNRTIDLTLSQRIRQFREMLISLVTAVDQLRNFYTHYHHSEIVIENKVLDFLNSSLVSTALHVKDKYLKTDKTKEFLKETIAAELDILIEAYKKKQIEKKNTRFKANKREDILNAIYNEAFWSFINDKDKDKETVVAKGADAYFEKNHHKSNDPDFALNISEKGIVYLLSFFLTNKEMDSLKANLTGFKGKVDRESGNSIKYMATQRIYSFHTYRGLKQKIRTSEEGVKETLLMQMIDELSKVPNVVYQHLSTTQQNSFIEDWNEYYKDYEDDVETDDLSRVIHPVIRKRYEDRFNYFAIRFLDEFFDFPTLRFQVHLGDYVHDRRTKQLGKVESDRIIKEKVTVFARLKDINSAKANYFHSLEEQDKEELDNKWTLFPNPSYDFPKEHTLQHQGEQKNAGKIGIYVKLRDTQYKEKAALEEARKSLNPKERSATKASKYDIITQIIEANDNVKSEKPLVFTGQPIAYLSMNDIHSMLFSLLTDNAELKKTPEEVEAKLIDQIGKQINEILSKDTDTKILKKYKDNDLKETDTDKITRDLARDKEEIEKLILEQKQRADDYNYTSSTKFNIDKSRKRKHLLFNAEKGKIGVWLANDIKRFMTEEFKSKWKGYQHTELQKLFAYYDTSKSDLDLILSDMVMVKDYPIELIALVKKSRTLVDFLNKYLEARLGYMENVITRVKNSIGTPQFKTVRKECFTFLKKSNYTVVSLDKQVERILSMPLFIERGFMDDKPTMLEGKSYQQHKEKFADWFVHYKENSNYQNFYDTEVYEITTEDKREKAKVTKKIKQQQKNDVFTLMMVNYMLEEVLKLSSNDRLSLNELYQTKEERIVNKQVAKDTQERNKNYIWNKVVDLQLCEGLVRIDKVKLKDIGNFRKYENDSRVKEFLTYQSDIVWSAYLSNEVDSNKLYVIERQLDNYESIRSKELLKEVQEIECSVYNQVANKESLKQSGNENFKQYVLQGLVPIGMDVREMLILSTDVKFIKEEIIQLGQAGEVEQDLYSLIYIRNKFAHNQLPIKEFFDFCENNYRSISDNEYYAEYYMEIFRSIKEKYTS (SEQ ID NO: 77) Myroides WP_MKDILTTDTTEKQNRFYSHKIADKYFFGGYFNLASNNIYEVFEE odoratimimus 006265509VNKRNTFGKLAKRDNGNLKNYIIHVFKDELSISDFEKRVAIFASYFPILETVDKKSIKERNRTIDLTLSQRIRQFREMLISLVTAVDQLRNFYTHYHHSEIVIENKVLDFLNSSLVSTALHVKDKYLKTDKTKEFLKETIAAELDILIEAYKKKQIEKKNTRFKANKREDILNAIYNEAFWSFINDKDKDKETVVAKGADAYFEKNHHKSNDPDFALNISEKGIVYLLSFFLTNKEMDSLKANLTGFKGKVDRESGNSIKYMATQRIYSFHTYRGLKQKIRTSEEGVKETLLMQMIDELSKVPNVVYQHLSTTQQNSFIEDWNEYYKDYEDDVETDDLSRVIHPVIRKRYEDRFNYFAIRFLDEFFDFPTLRFQVHLGDYVHDRRTKQLGKVESDRIIKEKVTVFARLKDINSAKASYFHSLEEQDKEELDNKWTLFPNPSYDFPKEHTLQHQGEQKNAGKIGIYVKLRDTQYKEKAALEEARKSLNPKERSATKASKYDIITQIIEANDNVKSEKPLVFTGQPIAYLSMNDIHSMLFSLLTDNAELKKTPEEVEAKLIDQIGKQINEILSKDTDTKILKKYKDNDLKETDTDKITRDLARDKEEIEKLILEQKQRADDYNYTSSTKFNIDKSRKRKHLLFNAEKGKIGVWLANDIKRFMFKESKSKWKGYQHTELQKLFAYFDTSKSDLELILSDMVMVKDYPIELIDLVRKSRTLVDFLNKYLEARLGYIENVITRVKNSIGTPQFKTVRKECFAFLKESNYTVASLDKQIERILSMPLFIERGFMDSKPTMLEGKSYQQHKEDFADWFVHYKENSNYQNFYDTEVYEIITEDKREQAKVTKKIKQQQKNDVFTLMMVNYMLEEVLKLPSNDRLSLNELYQTKEERIVNKQVAKDTQERNKNYIWNKVVDLQLCEGLVRIDKVKLKDIGNFRKYENDSRVKEFLTYQSDIVWSGYLSNEVDSNKLYVIERQLDNYESIRSKELLKEVQEIECIVYNQVANKESLKQSGNENFKQYVLQGLLPRGTDVREMLILSTDVKFKKEEIMQLGQVREVEQDLYSLIYIRNKFAHNQLPIKEFFDFCENNYRPISDNEYYAEYYMEIFRSIKEKYAS (SEQ ID NO: 78) Prevotella WP_MQKQDKLFVDRKKNAIFAFPKYITIMENQEKPEPIYYELTDKHF sp. MSX73 700412163WAAFLNLARHNVYTTINHINRRLEIAELKDDGYMMGIKGSWNEQAKKLDKKVRLRDLIMKHFPFLEAAAYEITNSKSPNNKEQREKEQSEALSLNNLKNVLFIFLEKLQVLRNYYSHYKYSEESPKPIFETSLLKNMYKVFDANVRLVKRDYMHHENIDMQRDFTHLNRKKQVGRTKNIIDSPNFHYHFADKEGNMTIAGLLFFVSLFLDKKDAIWMQKKLKGFKDGRNLREQMTNEVFCRSRISLPKLKLENVQTKDWMQLDMLNELVRCPKSLYERLREKDRESFKVPFDIFSDDYDAEEEPFKNTLVRHQDRFPYFVLRYFDLNEIFEQLRFQIDLGTYHFSIYNKRIGDEDEVRHLTHHLYGFARIQDFAPQNQPEEWRKLVKDLDHFETSQEPYISKTAPHYHLENEKIGIKFCSTHNNLFPSLKREKTCNGRSKFNLGTQFTAEAFLSVHELLPMMFYYLLLTKDYSRKESADKVEGIIRKEISNIYAIYDAFANNEINSIADLTCRLQKTNILQGHLPKQMISILEGRQKDMEKEAERKIGEMIDDTQRRLDLLCKQTNQKIRIGKRNAGLLKSGKIADWLVSDMMRFQPVQKDTNNAPINNSKANSTEYRMLQHALALFGSESSRLKAYFRQMNLVGNANPHPFLAETQWEHQTNILSFYRNYLEARKKYLKGLKPQNWKQYQHFLILKVQKTNRNTLVTGWKNSFNLPRGIFTQPIREWFEKHNNSKRIYDQILSFDRVGFVAKAIPLYFAEEYKDNVQPFYDYPFNIGNKLKPQKGQFLDKKERVELWQKNKELFKNYPSEKNKTDLAYLDFLSWKKFERELRLIKNQDIVTWLMFKELFKTTTVEGLKIGEIHLRDIDTNTANEESNNILNRIMPMKLPVKTYETDNKGNILKERPLATFYIEETETKVLKQGNFKVLAKDRRLNGLLSFAETTDIDLEKNPITKLSVDYELIKYQTTRISIFEMTLGLEKKLIDKYSTLPTDSFRNMLERWLQCKANRPELKNYVNSLIAVRNAFSHNQYPMYDATLFAEVKKFTLFPSVDTKKIELNIAPQLLEIVGKAIKEIEKSENKN (SEQ ID NO: 79) Porphyromonas WP_MTEQNERPYNGTYYTLEDKHFWAAFFNLARHNAYITLAHIDRQ gingivalis 201458414LAYSKADITNDEDILFFKGQWKNLDNDLERKARLRSLILKHFSFLEGAAYGKKLFESQSSGNKSSKKKELTKKEKEELQANALSLDNLKSILFDFLQKLKDFRNYYSHYRHPESSELPLFDGNMLQRLYNVFDVSVQRVKRDHEHNDKVDPHRHFNHLVRKGKKDRYGNNDNPFFKHHFVDREEKVTEAGLLFFVSLFLEKRDAIWMQKKIRGFKGGTETYQQMTNEVFCRSRISLPKLKLESLRTDDWMLLDMLNELVRCPKSLYDRLREEDRARFRVPVDILSDEDDTDGTEEDPFKNTLVRHQDRFPYFALRYFDLKKVFTSLRFHIDLGTYHFAIYKKNIGEQPEDRHLTRNLYGFGRIQDFAEEHRPEEWKRLVRDLDYFETGDKPYITQTTPHYHIEKGKIGLRFVPEGQHLWPSPEVGATRTGRSKYAQDKRLTAEAFLSVHELMPMMFYYFLLREKYSDEASAERVQGRIKRVIEDVYAVYDAFARGEINTRDELDACLADKGIRRGHLPRQMIGILSQEHKDMEEKVRKKLQEMIVDTDHRLDMLDRQTDRKIRIGRKNAGLPKSGVIADWLVRDMMRFQPVAKDTSGKPLNNSKANSTEYRMLQRALALEGGEKERLTPYFRQMNLTGGNNPHPFLHETRWESHTNILSFYRSYLKARKAFLQSIGRSDRVENHRFLLLKEPKTDRQTLVAGWKGEFHLPRGIFTEAVRDCLIEMGLDEVGSYKEVGFMAKAVPLYFERACKDRVQPFYDYPFNVGNSLKPKKGRFLSKEKRAEEWESGKERFRLAKLKKEILEAKEHPYLDFKSWQKFERELRLVKNQDIITWMICRDLMEENKVEGLDTGTLYLKDIRTDVQEQGNLNVLNRVKPMRLPVVVYRADSRGHVHKEQAPLATVYIEERDTKLLKQGNFKSFVKDRRLNGLFSFVDTGALAMEQYPISKLRVEYELAKYQTARVCAFEQTLELEESLLTRYPHLPDKNFRKMLESWSDPLLDKWPDLHGNVRLLIAVRNAFSHNQYPMYDEAVFSSIRKYDPSSPDAIEERMGLNIAHRLSEEVKQAKEMAERIIQA (SEQ ID NO: 80) PaludibacterWP_ MKTSANNIYENGINSFKKIFDSKGAIAPIAEKSCRNFDIKAQNDV propionicigenes013446107 NKEQRIHYFAVGHTFKQLDTENLFEYVLDENLRAKRPTRFISLQQFDKEFIENIKRLISDIRNINSHYIHRFDPLKIDAVPTNIIDFLKESFELAVIQIYLKEKGINYLQFSENPHADQKLVAFLHDKFLPLDEKKTSMLQNETPQLKEYKEYRKYFKTLSKQAAIDQLLFAEKETDYIWNLFDSHPVLTISAGKYLSFYSCLFLLSMFLYKSEANQLISKIKGFKKNTTEEEKSKREIFTFFSKRFNSMDIDSEENQLVKFRDLILYLNHYPVAWNKDLELDSSNPAMTDKLKSKIIELEINRSFPLYEGNERFATFAKYQIWGKKHLGKSIEKEYINASFTDEEITAYTYETDTCPELKDAHKKLADLKAAKGLFGKRKEKNESDIKKTETSIRELQHEPNPIKDKLIQRIEKNLLTVSYGRNQDRFMDFSARFLAEINYFGQDASFKMYHFYATDEQNSELEKYELPKDKKKYDSLKFHQGKLVHFISYKEHLKRYESWDDAFVIENNAIQLKLSFDGVENTVTIQRALLIYLLEDALRNIQNNTAENAGKQLLQEYYSHNKADLSAFKQILTQQDSIEPQQKTEFKKLLPRRLLNNYSPAINHLQTPHSSLPLILEKALLAEKRYCSLVVKAKAEGNYDDFIKRNKGKQFKLQFIRKAWNLMYFRNSYLQNVQAAGHHKSFHIERDEFNDFSRYMFAFEELSQYKYYLNEMFEKKGFFENNEFKILFQSGTSLENLYEKTKQKFEIWLASNTAKTNKPDNYHLNNYEQQFSNQLFFINLSHFINYLKSTGKLQTDANGQIIYEALNNVQYLIPEYYYTDKPERSESKSGNKLYNKLKATKLEDALLYEMAMCYLKADKQIADKAKHPITKLLTSDVEFNITNKEGIQLYHLLVPFKKIDAFIGLKMHKEQQDKKHPTSFLANIVNYLELVKNDKDIRKTYEAFSTNPVKRTLTYDDLAKIDGHLISKSIKFTNVTLELERYFIFKESLIVKKGNNIDFKYIKGLRNYYNNEKKKNEGIRNKAFHFGIPDSKSYDQLIRDAEVMFIANEVKPTHATKYTDLNKQLHTVCDKLMETVHNDYFSKEGDGKKKREAAGQ KYFENIISAK (SEQ ID NO: 81)Porphyromonas WP_ MTEQNEKPYNGTYYTLEDKHFWAAFFNLARHNAYITLAHIDRQ gingivalis013816155 LAYSKADITNDEDILFFKGQWKNLDNDLERKARLRSLILKHFSFLEGAAYGKKLFESQSSGNKSSKNKELTKKEKEELQANALSLDNLKSILFDFLQKLKDFRNYYSHYRHPESSELPLFDGNMLQRLYNVFDVSVQRVKRDHEHNDKVDPHRHFNHLVRKGKKDRYGNNDNPFFKHHFVDREGTVTEAGLLFFVSLFLEKRDAIWMQKKIRGFKGGTETYQQMTNEVFCRSRISLPKLKLESLRTDDWMLLDMLNELVRCPKSLYDRLREEDRARFRVPVDILSDEEDTDGAEEDPFKNTLVRHQDRFPYFALRYFDLKKVFTSLRFQIDLGTYHFAIYKKNIGEQPEDRHLTRNLYGFGRIQDFAEEHRPEEWKRLVRDLDYFETGDKPYITQTTPHYHIEKGKIGLRFVPEGQHLWPSPEVGATRTGRSKYAQDKRFTAEAFLSAHELMPMMFYYFLLREKYSEEASAERVQGRIKRVIEDVYAVYDAFARDEINTRDELDACLADKGIRRGHLPRQMIGILSQEHKDMEEKIRKKLQEMMADTDHRLDMLDRQTDRKIRIGRKNAGLPKSGVIADWLVRDMMRFQPVAKDTSGKPLNNSKANSTEYRMLQRALALFGGEKERLTPYFRQMNLTGGNNPHPFLHETRWESHTNILSFYRSYLKARKAFLQSIGRSDRVENHRFLLLKEPKTDRQTLVAGWKGEFHLPRGIFTEAVRDCLIEMGLDEVGSYKEVGFMAKAVPLYFERACKDWVQPFYNYPFNVGNSLKPKKGRFLSKEKRAEEWESGKERFRLAKLKKEILEAKEHPYLDFKSWQKFERELRLVKNQDIITWMICGDLMEENKVEGLDTGTLYLKDIRTDVQEQGSLNVLNRVKPMRLPVVVYRADSRGHVHKEQAPLATVYIEERDTKLLKQGNFKSFVKDRRLNGLFSFVDTGALAMEQYPISKLRVEYELAKYQTARVCAFEQTLELEESLLTRCPHLPDKNFRKMLESWSDPLLDKWPDLHRKVRLLIAVRNAFSHNQYPMYDEAVESSIRKYDPSFPDAIEERMGLNIAHRLSEEVKQAKETVERIIQA (SEQ ID NO: 82)Flavobacterium WP_ MSSKNESYNKQKTFNHYKQEDKYFFGGFLNNADDNLRQVGKE columnare014165541 FKTRINFNHNNNELASVFKDYFNKEKSVAKREHALNLLSNYFPVLERIQKHINHNFEQTREIFELLLDTIKKLRDYYTHHYHKPITINPKIYDFLDDTLLDVLITIKKKKVKNDTSRELLKEKLRPELTQLKNQKREELIKKGKKLLEENLENAVFNHCLRPFLEENKTDDKQNKTVSLRKYRKSKPNEETSITLTQSGLVFLMSFFLHRKEFQVFTSGLEGFKAKVNTIKEEEISLNKNNIVYMITHWSYSYYNFKGLKHRIKTDQGVSTLEQNNTTHSLTNTNTKEALLTQIVDYLSKVPNEIYETLSEKQQKEFEEDINEYMRENPENEDSTESSIVSHKVIRKRYENKFNYFAMRFLDEYAELPTLRFMVNFGDYIKDRQKKILESIQFDSERIIKKEIHLFEKLSLVTEYKKNVYLKETSNIDLSRFPLFPNPSYVMANNNIPFYIDSRSNNLDEYLNQKKKAQSQNKKRNLTFEKYNKEQSKDAIIAMLQKEIGVKDLQQRSTIGLLSCNELPSMLYEVIVKDIKGAELENKIAQKIREQYQSIRDFTLDSPQKDNIPTTLIKTINTDSSVTFENQPIDIPRLKNAIQKELTLTQEKLLNVKEHEIEVDNYNRNKNTYKFKNQPKNKVDDKKLQRKYVFYRNEIRQEANWLASDLIHFMKNKSLWKGYMHNELQSFLAFFEDKKNDCIALLETVFNLKEDCILTKGLKNLFLKHGNFIDFYKEYLKLKEDFLNTESTFLENGLIGLPPKILKKELSKRFKYIFIVFQKRQFIIKELEEKKNNLYADAINLSRGIFDEKPTMIPFKKPNPDEFASWFVASYQYNNYQSFYELTPDIVERDKKKKYKNLRAINKVKIQDYYLKLMVDTLYQDLFNQPLDKSLSDFYVSKAEREKIKADAKAYQKRNDSSLWNKVIHLSLQNNRITANPKLKDIGKYKRALQDEKIATLLTYDDRTWTYALQKPEKENENDYKELHYTALNMELQEYEKVRSKELLKQVQELEKQILEEYTDFLSTQIHPADFEREGNPNFKKYLAHSILENEDDLDKLPEKVEAMRELDETITNPIIKKAIVLIIIRNKMAHNQYPPKFIYDLANRFVPKKEEEYFATYFNRVFETITKELWENKEKKDKTQV (SEQ ID NO: 83) Psychroflexus WP_MESIIGLGLSFNPYKTADKHYFGSFLNLVENNLNAVFAEFKERIS torquis 015024765YKAKDENISSLIEKHFIDNMSIVDYEKKISILNGYLPIIDFLDDELENNLNTRVKNFKKNFIILAEAIEKLRDYYTHFYHDPITFEDNKEPLLELLDEVLLKTILDVKKKYLKTDKTKEILKDSLREEMDLLVIRKTDELREKKKTNPKIQHTDSSQIKNSIFNDAFQGLLYEDKGNNKKTQVSHRAKTRLNPKDIHKQEERDFEIPLSTSGLVFLMSLFLSKKEIEDFKSNIKGFKGKVVKDENHNSLKYMATHRVYSILAFKGLKYRIKTDTFSKETLMMQMIDELSKVPDCVYQNLSETKQKDFIEDWNEYFKDNEENTENLENSRVVHPVIRKRYEDKFNYFAIRFLDEFANFKTLKFQVFMGYYIHDQRIKTIGTTNITTERTVKEKINVFGKLSKMDNLKKHFFSQLSDDENTDWEFFPNPSYNFLTQADNSPANNIPIYLELKNQQIIKEKDAIKAEVNQTQNRNPNKPSKRDLLNKILKTYEDFHQGDPTAILSLNEIPALLHLFLVKPNNKTGQQIENIIRIKIEKQFKAINHPSKNNKGIPKSLFADTNVRVNAIKLKKDLEAELDMLNKKHIAFKENQKASSNYDKLLKEHQFTPKNKRPELRKYVFYKSEKGEEATWLANDIKRFMPKDFKTKWKGCQHSELQRKLAFYDRHTKQDIKELLSGCEFDHSLLDINAYFQKDNFEDFFSKYLENRIETLEGVLKKLHDFKNEPTPLKGVFKNCFKFLKRQNYVTESPEIIKKRILAKPTFLPRGVFDERPTMKKGKNPLKDKNEFAEWFVEYLENKDYQKFYNAEEYRMRDADFKKNAVIKKQKLKDFYTLQMVNYLLKEVFGKDEMNLQLSELFQTRQERLKLQGIAKKQMNKETGDSSENTRNQTYIWNKDVPVSFFNGKVTIDKVKLKNIGKYKRYERDERVKTFIGYEVDEKWMMYLPHNWKDRYSVKPINVIDLQIQEYEEIRSHELLKEIQNLEQYIYDHTTDKNILLQDGNPNFKMYVLNGLLIGIKQVNIPDFIVLKQNTNFDKIDFTGIASCSELEKKTIILIAIRNKFAHNQLPNKMIYDLANEFLKIEKNETYANYYLKVLKKMISD LA (SEQ ID NO: 84)Riemerella WP_ MFFSFHNAQRVIFKHLYKAFDASLRMVKEDYKAHFTVNLTRDF anatipestifer015345620 AHLNRKGKNKQDNPDFNRYRFEKDGFFTESGLLFFTNLFLDKRDAYWMLKKVSGFKASHKQREKMTTEVFCRSRILLPKLRLESRYDHNQMLLDMLSELSRCPKLLYEKLSEENKKHFQVEADGFLDEIEEEQNPFKDTLIRHQDRFPYFALRYLDLNESFKSIRFQVDLGTYHYCIYDKKIGDEQEKRHLTRTLLSFGRLQDFTEINRPQEWKALTKDLDYKETSNQPFISKTTPHYHITDNKIGFRLGTSKELYPSLEIKDGANRIAKYPYNSGFVAHAFISVHELLPLMFYQHLTGKSEDLLKETVRHIQRIYKDFEEERINTIEDLEKANQGRLPLGAFPKQMLGLLQNKQPDLSEKAKIKIEKLIAETKLLSHRLNTKLKSSPKLGKRREKLIKTGVLADWLVKDFMRFQPVAYDAQNQPIKSSKANSTEFWFIRRALALYGGEKNRLEGYFKQTNLIGNTNPHPFLNKFNWKACRNLVDFYQQYLEQREKFLEAIKHQPWEPYQYCLLLKVPKENRKNLVKGWEQGGISLPRGLFTEAIRETLSKDLTLSKPIRKEIKKHGRVGFISRAITLYFKEKYQDKHQSFYNLSYKLEAKAPLLKKEEHYEYWQQNKPQSPTESQRLELHTSDRWKDYLLYKRWQHLEKKLRLYRNQDIMLWLMTLELTKNHFKELNLNYHQLKLENLAVNVQEADAKLNPLNQTLPMVLPVKVYPTTAFGEVQYHETPIRTVYIREEQTKALKMGNFKALVKDRRLNGLFSFIKEENDTQKHPISQLRLRRELEIYQSLRVDAFKETLSLEEKLLNKHASLSSLENEFRTLLEEWKKKYAASSMVTDKHIAFIASVRNAFCHNQYPFYKETLHAPILLFTVAQPTTEEKDGLGIAEALLKVLREYCEIVKSQI (SEQ ID NO: 85) Prevotella WP_MENDKRLEESACYTLNDKHFWAAFLNLARHNVYITVNHINKTL pleuritidis 021584635ELKNKKNQEIIIDNDQDILAIKTHWAKVNGDLNKTDRLRELMIKHFPFLEAAIYSNNKEDKEEVKEEKQAKAQSFKSLKDCLFLFLEKLQEARNYYSHYKYSESSKEPEFEEGLLEKMYNTFDASIRLVKEDYQYNKDIDPEKDFKHLERKEDFNYLFTDKDNKGKITKNGLLFFVSLFLEKKDAIWMQQKFRGFKDNRGNKEKMTHEVFCRSRMLLPKIRLESTQTQDWILLDMLNELIRCPKSLYERLQGAYREKFKVPFDSIDEDYDAEQEPFRNTLVRHQDRFPYFALRYFDYNEIFKNLRFQIDLGTYHFSIYKKLIGGKKEDRHLTHKLYGFERIQEFTKQNRPDKWQAIIKDLDTYETSNERYISETTPHYHLENQKIGIRFRNDNNDIWPSLKTNGEKNEKSKYNLDKPYQAEAFLSVHELLPMMFYYLLLKMENTDNDKEDNEVGTKKKGNKNNKQEKHKIEEIIENKIKDIYALYDAFTNGEINSIDELAEQREGKDIEIGHLPKQLIVILKNKSKDMAEKANRKQKEMIKDTKKRLATLDKQVKGEIEDGGRNIRLLKSGEIARWLVNDMMRFQPVQKDNEGKPLNNSKANSTEYQMLQRSLALYNKEEKPTRYFRQVNLIKSSNPHPFLEDTKWEECYNILSFYRNYLKAKIKFLNKLKPEDWKKNQYFLMLKEPKTNRKTLVQGWKNGFNLPRGIFTEPIKEWFKRHQNDSEEYKKVEALDRVGLVAKVIPLFFKEEYFKEDAQKEINNCVQPFYSFPYNVGNIHKPEEKNFLHCEERRKLWDKKKDKFKGYKAKEKSKKMTDKEKEEHRSYLEFQSWNKFERELRLVRNQDILTWLLCTKLIDKLKIDELNIEELQKLRLKDIDTDTAKKEKNNILNRVMPMRLPVTVYEIDKSFNIVKDKPLHTVYIEETGTKLLKQGNFKALVKDRRLNGLFSFVKTSSEAESKSKPISKLRVEYELGAYQKARIDIIKDMLALEKTLIDNDENLPTNKFSDMLKSWLKGKGEANKARLQNDVGLLVAVRNAFSHNQYPMYNSEVFKGMKLLSLSSDIPEKEGLGIAKQLKDKIKETIERIIEIEKE IRN (SEQ ID NO: 86)Porphyromonas WP_ MNTVPASENKGQSRTVEDDPQYFGLYLNLARENLIEVESHVRIKgingivalis 021663197 FGKKKLNEESLKQSLLCDHLLSVDRWTKVYGHSRRYLPFLHYFDPDSQIEKDHDSKTGVDPDSAQRLIRELYSLLDFLRNDFSHNRLDGTTFEHLEVSPDISSFITGTYSLACGRAQSRFADFFKPDDFVLAKNRKEQLISVADGKECLTVSGLAFFICLFLDREQASGMLSRIRGFKRTDENWARAVHETFCDLCIRHPHDRLESSNTKEALLLDMLNELNRCPRILYDMLPEEERAQFLPALDENSMNNLSENSLNEESRLLWDGSSDWAEALTKRIRHQDRFPYLMLRFIEEMDLLKGIRFRVDLGEIELDSYSKKVGRNGEYDRTITDHALAFGKLSDFQNEEEVSRMISGEASYPVRFSLFAPRYAIYDNKIGYCHTSDPVYPKSKTGEKRALSNPRSMGFISVHDLRKLLLMELLCEGSFSRMQSDFLRKANRILDETAEGKLQFSALFPEMRHRFIPPQNPKSKDRREKAETTLEKYKQEIKGRKDKLNSQLLSAFDMDQRQLPSRLLDEWMNIRPASHSVKLRTYVKQLNEDCRLRLQKFRKDGDGKARAIPLVGEMATFLSQDIVRMIISEETKKLITSAYYNEMQRSLAQYAGEENRHQFRAIVAELRLLDPSSGHPFLSATMETAHRYTEDFYKCYLEKKREWLAKTFYRPEQDENTKRRISVFFVPDGEARKLLPLLIRRRMKEQNDLQDWIRNKQAHPIDLPSHLFDSKIMELLKVKDGKKKWNEAFKDWWSTKYPDGMQPFYGLRRELNIHGKSVSYIPSDGKKFADCYTHLMEKTVQDKKRELRTAGKPVPPDLAADIKRSFHRAVNEREFMLRLVQEDDRLMLMAINKMMTDREEDILPGLKNIDSILDEENQFSLAVHAKVLEKEGEGGDNSLSLVPATIEIKSKRKDWSKYIRYRYDRRVPGLMSHFPEHKATLDEVKTLLGEYDRCRIKIFDWAFALEGAIMSDRDLKPYLHESSSREGKSGEHSTLVKMLVEKKGCLTPDESQYLILIRNKAAHNQFPCAAEMPLIYRDVSAKVGSIEGSSAKDLPEGSSLVDSLWKKYEMIIRKILPILDPENRFFGKLLNNMSQPINDL (SEQ ID NO: 87)Porphyromonas WP_ MNTVPASENKGQSRTVEDDPQYFGLYLNLARENLIEVESHVRIKgingivalis 021665475 FGKKKLNEESLKQSLLCDHLLSVDRWTKVYGHSRRYLPFLHYFDPDSQIEKDHDSKTGVDPDSAQRLIRELYSLLDFLRNDFSHNRLDGTTFEHLEVSPDISSFITGTYSLACGRAQSRFADFFKPDDFVLAKNRKEQLISVADGKECLTVSGLAFFICLFLDREQASGMLSRIRGFKRTNENWARAVHETFCDLCIRHPHDRLESSNTKEALLLDMLNELNRCPRILYDMLPEEERAQFLPALDENSMNNLSENSLNEESRLLWDGSSDWAEALTKRIRHQDRFPYLMLRFIEEMDLLKGIRFRVDLGEIELDSYSKKVGRNGEYDRTITDHALAFGKLSDFQNEEEVSRMISGEASYPVRFSLFAPRYAIYDNKIGYCHTSDPVYPKSKTGEKRALSNPQSMGFISVHDLRKLLLMELLCEGSFSRMQSGFLRKANRILDETAEGKLQFSALFPEMRHRFIPPQNPKSKDRREKAETTLEKYKQEIKGRKDKLNSQLLSAFDMNQRQLPSRLLDEWMNIRPASHSVKLRTYVKQLNEDCRLRLRKFRKDGDGKARAIPLVGEMATFLSQDIVRMIISEETKKLITSAYYNEMQRSLAQYAGEENRRQFRAIVAELHLLDPSSGHPFLSATMETAHRYTEDFYKCYLEKKREWLAKTFYRPEQDENTKRRISVFFVPDGEARKLLPLLIRRRMKEQNDLQDWIRNKQAHPIDLPSHLFDSKIMELLKVKDGKKKWNEAFKDWWSTKYPDGMQPFYGLRRELNIHGKSVSYIPSDGKKFADCYTHLMEKTVQDKKRELRTAGKPVPPDLAADIKRSFHRAVNEREFMLRLVQEDDRLMLMAINKMMTDREEDILPGLKNIDSILDKENQFSLAVHAKVLEKEGEGGDNSLSLVPATIEIKSKRKDWSKYIRYRYDRRVPGLMSHFPEHKATLDEVKTLLGEYDRCRIKIFDWAFALEGAIMSDRDLKPYLHESSSREGKSGEHSTLVKMLVEKKGCLTPDESQYLILIRNKAAHNQFPCAAEMPLIYRDVSAKVGSIEGSSAKDLPEGSSLVDSLWKKYEMIIRKILPILDHENRFFGKLLNNMSQPINDL (SEQ ID NO: 88)Porphyromonas WP_ MNTVPASENKGQSRTVEDDPQYFGLYLNLARENLIEVESHVRIKgingivalis 021677657 FGKKKLNEESLKQSLLCDHLLSVDRWTKVYGHSRRYLPFLHYFDPDSQIEKDHDSKTGVDPDSAQRLIRELYSLLDFLRNDFSHNRLDGTTFEHLEVSPDISSFITGTYSLACGRAQSRFADFFKPDDFVLAKNRKEQLISVADGKECLTVSGLAFFICLFLDREQASGMLSRIRGFKRTDENWARAVHETFCDLCIRHPHDRLESSNTKEALLLDMLNELNRCPRILYDMLPEEERAQFLPALDENSMNNLSENSLNEESRLLWDGSSDWAEALTKRIRHQDRFPYLMLRFIEEMDLLKGIRFRVDLGEIELDSYSKKVGRNGEYDRTITDHALAFGKLSDFQNEEEVSRMISGEASYPVRFSLFAPRYAIYDNKIGYCHTSDPVYPKSKTGEKRALSNPQSMGFISVHDLRKLLLMELLCEGSFSRMQSGFLRKANRILDETAEGKLQFSALFPEMRHRFIPPQNPKSKDRREKAETTLEKYKQEIKGRKDKLNSQLLSAFDMNQRQLPSRLLDEWMNIRPASHSVKLRTYVKQLNEDCRLRLRKFRKDGDGKARAIPLVGEMATFLSQDIVRMIISEETKKLITSAYYNEMQRSLAQYAGEENRRQFRAIVAELHLLDPSSGHPFLSATMETAHRYTEDFYKCYLEKKREWLAKTFYRPEQDENTKRRISVFFVPDGEARKLLPLLIRRRMKEQNDLQDWIRNKQAHPIDLPSHLFDSKIMELLKVKDGKKKWNEAFKDWWSTKYPDGMQPFYGLRRELNIHGKSVSYIPSDGKKFADCYTHLMEKTVQDKKRELRTAGKPVPPDLAADIKRSFHRAVNEREFMLRLVQEDDRLMLMAINKMMTDREEDILPGLKNIDSILDEENQFSLAVHAKVLEKEGEGGDNSLSLVPATIEIKSKRKDWSKYIRYRYDRRVPGLMSHFPEHKATLDEVKTLLGEYDRCRIKIFDWAFALEGAIMSDRDLKPYLHESSSREGKSGEHSTLVKMLVEKKGCLTPDESQYLILIRNKAAHNQFPCAAEMPLIYRDVSAKVGSIEGSSAKDLPEGSSLVDSLWKKYEMIIRKILPILDHENRFFGKLLNNMSQPINDL (SEQ ID NO: 89)Porphyromonas WP_ MNTVPASENKGQSRTVEDDPQYFGLYLNLARENLIEVESHVRIKgingivalis 102680012 FGKKKLNEESLKQSLLCDHLLSVDRWTKVYGHSRRYLPFLHYFDPDSQIEKDHDSKTGVDPDSAQRLIRELYSLLDFLRNDFSHNRLDGTTFEHLEVSPDISSFITGTYSLACGRAQSRFADFFKPDDFVLAKNRKEQLISVADGKECLTVSGLAFFICLFLDREQASGMLSRIRGFKRTDENWARAVHETFCDLCIRHPHDRLESSNTKEALLLDMLNELNRCPRILYDMLPEEERAQFLPALDENSMNNLSENSLNEESRLLWDGSSDWAEALTKRIRHQDRFPYLMLRFIEEMDLLKGIRFRVDLGEIELDSYSKKVGRNGEYDRTITDHALAFGKLSDFQNEEEVSRMISGEASYPVRFSLFAPRYAIYDNKIGYCHTSDPVYPKSKTGEKRALSNPRSMGFISVHDLRKLLLMELLCEGSFSRMQSDFLRKANRILDETAEGKLQFSALFPEMRHRFIPPQNPKSKDRREKAETTLEKYKQEIKGRKDKLNSQLLSAFDMDQRQLPSRLLDEWMNIRPASHSVKLRTYVKQLNEDCRLRLQKFRKDGDGKARAIPLVGEMATFLSQDIVRMIISEETKKLITSAYYNEMQRSLAQYAGEENRHQFRAIVAELRLLDPSSGHPFLSATMETAHRYTEDFYKCYLEKKREWLAKTFYRPEQDENTKRRISVFFVPDGEARKLLPLLIRRRMKEQNDLQDWIRNKQAHPIDLPSHLFDSKVMELLKVKDGKKKWNEAFKDWWSTKYPDGMQPFYGLRRELNIHGKSVSYIPSDGKKFADCYTHLMEKTVRDKKRELRTAGKPVPPDLAAYIKRSFHRAVNEREFMLRLVQEDDRLMLMAINKIMTDREEDILPGLKNIDSILDKENQFSLAVHAKVLEKEGEGGDNSLSLVPATIEIKSKRKDWSKYIRYRYDRRVPGLMSHFPEHKATLDEVKTLLGEYDRCRIKIFDWAFALEGAIMSDRDLKPYLHESSSREGKSGEHSTLVKMLVEKKGCLTPDESQYLILIRNKAAHNQFPCAAEIPLIYRDVSAKVGSIEGSSAKDLPEGSSLVDSLWKKYEMIIRKILPILDPENRFFGKLLNNMSQPINDL (SEQ ID NO: 90)Porphyromonas WP_ MNTVPASENKGQSRTVEDDPQYFGLYLNLARENLIEVESHVRIKgingivalis 023846767 FGKKKLNEESLKQSLLCDHLLSVDRWTKVYGHSRRYLPFLHYFDPDSQIEKDHDSKTGVDPDSAQRLIRELYSLLDFLRNDFSHNRLDGTTFEHLEVSPDISSFITGTYSLACGRAQSRFADFFKPDDFVLAKNRKEQLISVADGKECLTVSGLAFFICLFLDREQASGMLSRIRGFKRTDENWARAVHETFCDLCIRHPHDRLESSNTKEALLLDMLNELNRCPRILYDMLPEEERAQFLPALDENSMNNLSENSLNEESRLLWDGSSDWAEALTKRIRHQDRFPYLMLRFIEEMDLLKGIRFRVDLGEIELDSYSKKVGRNGEYDRTITDHALAFGKLSDFQNEEEVSRMISGEASYPVRFSLFAPRYAIYDNKIGYCHTSDPVYPKSKTGEKRALSNPRSMGFISVHDLRKLLLMELLCEGSFSRMQSDFLRKANRILDETAEGKLQFSALFPEMRHRFIPPQNPKSKDRREKAETTLEKYKQEIKGRKDKLNSQLLSAFDMNQRQLPSRLLDEWMNIRPASHSVKLRTYVKQLNEDCRLRLRKFRKDGDGKARAIPLVGEMATFLSQDIVRMIISEETKKLITSAYYNEMQRSLAQYAGEENRRQFRAIVAELHLLDPSSGHPFLSATMETAHRYTEDFYKCYLEKKREWLAKTFYRPEQDENTKRRISVFFVPDGEARKLLPLLIRRRMKEQNDLQDWIRNKQAHPIDLPSHLFDSKIMELLKVKDGKKKWNEAFKDWWSTKYPDGMQPFYGLRRELNIHGKSVSYIPSDGKKFADCYTHLMEKTVQDKKRELRTAGKPVPPDLAADIKRSFHRAVNEREFMLRLVQEDDRLMLMAINKMMTDREEDILPGLKNIDSILDEENQFSLAVHAKVLEKEGEGGDNSLSLVPATIEIKSKRKDWSKYIRYRYDRRVPGLMSHFPEHKATLDEVKTLLGEYDRCRIKIFDWAFALEGAIMSDRDLKPYLHESSSREGKSGEHSTLVKMLVEKKGCLTPDESQYLILIRNKAAHNQFPCAAEMPLIYRDVSAKVGSIEGSSAKDLPEGSSLVDSLWKKYEMIIRKILPILDPENRFFGKLLNNMSQPINDL (SEQ ID NO: 91) PrevotellaWP_ MKNDNNSTKSTDYTLGDKHFWAAFLNLARHNVYITVNHINKV falsenii 036884929LELKNKKDQEIIIDNDQDILAIKTLWGKVDTDINKKDRLRELIMKHFPFLEAATYQQSSTNNTKQKEEEQAKAQSFESLKDCLFLFLEKLREARNYYSHYKHSKSLEEPKLEEKLLENMYNIFDTNVQLVIKDYEHNKDINPEEDFKHLGRAEGEFNYYFTRNKKGNITESGLLFFVSLFLEKKDAIWAQTKIKGFKDNRENKQKMTHEVFCRSRMLLPKLRLESTQTQDWILLDMLNELIRCPKSLYKRLQGEKREKFRVPFDPADEDYDAEQEPFKNTLVRHQDRFPYFALRYFDYNEIFTNLRFQIDLGTYHFSIYKKQIGDKKEDRHLTHKLYGFERIQEFAKENRPDEWKALVKDLDTFEESNEPYISETTPHYHLENQKIGIRNKNKKKKKTIWPSLETKTTVNERSKYNLGKSFKAEAFLSVHELLPMMFYYLLLNKEEPNNGKINASKVEGIIEKKIRDIYKLYGAFANEEINNEEELKEYCEGKDIAIRHLPKQMIAILKNEYKDMAKKAEDKQKKMIKDTKKRLAALDKQVKGEVEDGGRNIKPLKSGRIASWLVNDMMRFQPVQRDRDGYPLNNSKANSTEYQLLQRTLALFGSERERLAPYFRQMNLIGKDNPHPFLKDTKWKEHNNILSFYRSYLEAKKNFLGSLKPEDWKKNQYFLKLKEPKTNRETLVQGWKNGFNLPRGIFTEPIREWFIRHQNESEEYKKVKDFDRIGLVAKVIPLFFKEDYQKEIEDYVQPFYGYPFNVGNIHNSQEGTFLNKKEREELWKGNKTKFKDYKTKEKNKEKTNKDKFKKKTDEEKEEFRSYLDFQSWKKFERELRLVRNQDIVTWLLCMELIDKLKIDELNIEELQKLRLKDIDTDTAKKEKNNILNRIMPMELPVTVYETDDSNNIIKDKPLHTIYIKEAETKLLKQGNFKALVKDRRLNGLFSFVETSSEAELKSKPISKSLVEYELGEYQRARVEIIKDMLRLEETLIGNDEKLPTNKFRQMLDKWLEHKKETDDTDLKNDVKLLTEVRNAFSHNQYPMRDRIAFANIKPFSLSSANTSNEEGLGIAKKLKDKTKETIDRIIEIEEQTATKR (SEQ ID NO: 92) PrevotellaWP_ MENDKRLEESTCYTLNDKHFWAAFLNLARHNVYITINHINKLL pleuritidis 036931485EIRQIDNDEKVLDIKALWQKVDKDINQKARLRELMIKHFPFLEAAIYSNNKEDKEEVKEEKQAKAQSFKSLKDCLFLFLEKLQEARNYYSHYKSSESSKEPEFEEGLLEKMYNTFGVSIRLVKEDYQYNKDIDPEKDFKHLERKEDFNYLFTDKDNKGKITKNGLLFFVSLFLEKKDAIWMQQKLRGFKDNRGNKEKMTHEVFCRSRMLLPKIRLESTQTQDWILLDMLNELIRCPKSLYERLQGAYREKFKVPFDSIDEDYDAEQEPFRNTLVRHQDRFPYFALRYFDYNEIFKNLRFQIDLGTYHFSIYKKLIGDNKEDRHLTHKLYGFERIQEFAKQKRPNEWQALVKDLDIYETSNEQYISETTPHYHLENQKIGIRFKNKKDKIWPSLETNGKENEKSKYNLDKSFQAEAFLSIHELLPMMFYDLLLKKEEPNNDEKNASIVEGFIKKEIKRMYAIYDAFANEEINSKEGLEEYCKNKGFQERHLPKQMIAILTNKSKNMAEKAKRKQKEMIKDTKKRLATLDKQVKGEIEDGGRNIRLLKSGEIARWLVNDMMRFQSVQKDKEGKPLNNSKANSTEYQMLQRSLALYNKEQKPTPYFIQVNLIKSSNPHPFLEETKWEECNNILSFYRSYLEAKKNFLESLKPEDWKKNQYFLMLKEPKTNRKTLVQGWKNGFNLPRGIFTEPIKEWFKRHQNDSEEYKKVEALDRVGLVAKVIPLFFKEEYFKEDAQKEINNCVQPFYSFPYNVGNIHKPEEKNFLHCEERRKLWDKKKDKFKGYKAKEKSKKMTDKEKEEHRSYLEFQSWNKFERELRLVRNQDIVTWLLCTELIDKLKIDELNIEELQKLRLKDIDTDTAKKEKNNILNRIMPMQLPVTVYEIDKSFNIVKDKPLHTIYIEETGTKLLKQGNFKALVKDRRLNGLFSFVKTSSEAESKSKPISKLRVEYELGAYQKARIDIIKDMLALEKTLIDNDENLPTNKFSDMLKSWLKGKGEANKARLQNDVDLLVAIRNAFSHNQYPMYNSEVFKGMKLLSLSSDIPEKEGLGIAKQLKDKIKETIERIIEIEKEIRN (SEQ ID NO: 93) [Porphyromonas WP_MTEQNERPYNGTYYTLEDKHFWAAFFNLARHNAYITLAHIDRQ gingivalis 039417390LAYSKADITNDEDILFFKGQWKNLDNDLERKARLRSLILKHFSFLEGAAYGKKLFESQSSGNKSSKKKELTKKEKEELQANALSLDNLKSILFDFLQKLKDFRNYYSHYRHPESSELPLFDGNMLQRLYNVFDVSVQRVKRDHEHNDKVDPHRHFNHLVRKGKKDRYGNNDNPFFKHHFVDREGTVTEAGLLFFVSLFLEKRDAIWMQKKIRGFKGGTEAYQQMTNEVFCRSRISLPKLKLESLRTDDWMLLDMLNELVRCPKSLYDRLREEDRARFRVPIDILSDEDDTDGTEEDPFKNTLVRHQDRFPYFALRYFDLKKVFTSLRFHIDLGTYHFAIYKKNIGEQPEDRHLTRNLYGFGRIQDFAEEHRPEEWKRLVRDLDYFETGDKPYITQTTPHYHIEKGKIGLRFVPEGQHLWPSPEVGATRTGRSKYAQDKRLTAEAFLSVHELMPMMFYYFLLREKYSEEVSAEKVQGRIKRVIEDVYAVYDAFARGEIDTLDRLDACLADKGIRRGHLPRQMIAILSQEHKDMEEKVRKKLQEMIADTDHRLDMLDRQTDRKIRIGRKNAGLPKSGVIADWLVRDMMRFQPVAKDTSGKPLNNSKANSTEYRMLQRALALFGGEKERLTPYFRQMNLTGGNNPHPFLHETRWESHTNILSFYRSYLKARKAFLQSIGRSDREENHRFLLLKEPKTDRQTLVAGWKSEFHLPRGIFTEAVRDCLIEMGYDEVGSYKEVGFMAKAVPLYFERACKDRVQPFYDYPFNVGNSLKPKKGRFLSKEKRAEEWESGKERFRLAKLKKEILEAKEHPYLDFKSWQKFERELRLVKNQDIITWMMCRDLMEENKVEGLDTGTLYLKDIRTDVHEQGSLNVLNRVKPMRLPVVVYRADSRGHVHKEQAPLATVYIEERDTKLLKQGNFKSFVKDRRLNGLFSFVDTGALAMEQYPISKLRVEYELAKYQTARVCAFEQTLELEESLLTRYPHLPDKNFRKMLESWSDPLLDKWPDLHRKVRLLIAVRNAFSHNQYPMYDEAVESSIRKYDPSSPDAIEERMGLNIAHRLSEEVKQAKEMAERIIQV (SEQ ID NO: 94) PorphyromonasWP_ MTEQSERPYNGTYYTLEDKHFWAAFLNLARHNAYITLTHIDRQ gulae 039418912LAYSKADITNDQDVLSFKALWKNLDNDLERKSRLRSLILKHFSFLEGAAYGKKLFESKSSGNKSSKNKELTKKEKEELQANALSLDNLKSILFDFLQKLKDFRNYYSHYRHSGSSELPLFDGNMLQRLYNVFDVSVQRVKRDHEHNDKVDPHRHFNHLVRKGKKDRYGHNDNPSFKHHFVDSEGMVTEAGLLFFVSLFLEKRDAIWMQKKIRGFKGGTETYQQMTNEVFCRSRISLPKLKLESLRMDDWMLLDMLNELVRCPKPLYDRLREDDRACFRVPVDILPDEDDTDGGGEDPFKNTLVRHQDRFPYFALRYFDLKKVFTSLRFHIDLGTYHFAIYKKMIGEQPEDRHLTRNLYGFGRIQDFAEEHRPEEWKRLVRDLDYFETGDKPYISQTSPHYHIEKGKIGLRFMPEGQHLWPSPEVGTTRTGRSKYAQDKRLTAEAFLSVHELMPMMFYYFLLREKYSEEVSAEKVQGRIKRVIEDVYAIYDAFARDEINTLKELDACLADKGIRRGHLPKQMIAILSQEHKNMEEKVRKKLQEMIADTDHRLDMLDRQTDRKIRIGRKNAGLPKSGVIADWLVRDMMRFQPVAKDASGKPLNNSKANSTEYRMLQRALALFGGEKERLTPYFRQMNLTGGNNPHPFLHDTRWESHTNILSFYRSYLRARKAFLERIGRSDRMENRPFLLLKEPKTDRQTLVAGWKSEFHLPRGIFTEAVRDCLIEMGYDEVGSYREVGFMAKAVPLYFERACEDRVQPFYDSPFNVGNSLKPKKGRFLSKEERAEEWERGKERFRDLEAWSHSAARRIEDAFAGIEYASPGNKKKIEQLLRDLSLWEAFESKLKVRADKINLAKLKKEILEAQEHPYHDFKSWQKFERELRLVKNQDIITWMMCRDLMEENKVEGLDTGTLYLKDIRTNVQEQGSLNVLNHVKPMRLPVVVYRADSRGHVHKEEAPLATVYIEERDTKLLKQGNFKSFVKDRRLNGLFSFVDTGGLAMEQYPISKLRVEYELAKYQTARVCAFEQTLELEESLLTRYPHLPDKNFRKMLESWSDPLLAKWPELHGKVRLLIAVRNAFSHNQYPMYDEAVESSIRKYDPSSPDAIEERMGLNIAHRLSEEVKQAK ETVERIIQA (SEQ ID NO: 95)Porphyromonas WP_ MTEQSERPYNGTYYTLEDKHFWAAFLNLARHNAYITLTHIDRQ gulae039419792 LAYSKADITNDQDVLSFKALWKNLDNDLERKSRLRSLILKHFSFLEGAAYGKKLFESKSSGNKSSKNKELTKKEKEELQANALSLDNLKSILFDFLQKLKDFRNYYSHYRHSGSSELPLFDGNMLQRLYNVFDVSVQRVKRDHEHNDKVDPHRHFNHLVRKGKKDRYGHNDNPSFKHHFVDGEGMVTEAGLLFFVSLFLEKRDAIWMQKKIRGFKGGTETYQQMTNEVFCRSRISLPKLKLESLRTDDWMLLDMLNELVRCPKPLYDRLREKDRARFRVPVDILPDEDDTDGGGEDPFKNTLVRHQDRFPYFALRYFDLKKVFTSLRFHIDLGTYHFAIYKKVIGEQPEDRHLTRNLYGFGRIQDFAEEHRPEEWKRLVRDLDYFETGDKPYISQTTPHYHIEKGKIGLRFVPEGQHLWPSPEVGTTRTGRSKYAQDKRLTAEAFLSVHELMPMMFYYFLLREKYSEEVSAEKVQGRIKRVIEDVYAIYDAFARDEINTRDELDACLADKGIRRGHLPKQMIGILSQEHKNMEEKVRKKLQEMIADTDHRLDMLDRQTDRKIRIGRKNAGLPKSGVIADWLVRDMMRFQPVAKDTSGKPLNNSKANSTEYRMLQRALALFGGEKERLTPYFRQMNLTGGNNPHPFLDETRWESHTNILSFYRSYLRARKAFLERIGRSDRVENRPFLLLKEPKTDRQTLVAGWKSEFHLPRGIFTEAVRDCLIEMGYDEVGSYKEVGFMAKAVPLYFERACKDRVQPFYDSPFNVGNSLKPKKGRFLSKEKRAEEWESGKERFRLAKLKKEILEAQEHPYHDFKSWQKFERELRLVKNQDIITWMMCRDLMEENKVEGLDTGTLYLKDIRPNVQEQGSLNVLNRVKPMRLPVVVYRADSRGHVHKEEAPLATVYIEERDTKLLKQGNFKSFVKDRRLNGLFSFVDTGGLAMEQYPISKLRVEYELAKYQTARVCVFELTLRLEESLLSRYPHLPDESFREMLESWSDPLLAKWPELHGKVRLLIAVRNAFSHNQYPMYDEAVFSSIRKYDPSSPDAIEERMGLNIAHRLSEEVKQAKETVERIIQA (SEQ ID NO: 96) PorphyromonasWP_ MTEQSERPYNGTYYTLEDKHFWAAFLNLARHNAYITLTHIDRQ gulae 039426176LAYSKADITNDQDVLSFKALWKNFDNDLERKSRLRSLILKHFSFLEGAAYGKKLFESKSSGNKSSKNKELTKKEKEELQANALSLDNLKSILFDFLQKLKDFRNYYSHYRHSGSSELPLFDGNMLQRLYNVFDVSVQRVKRDHEHNDKVDPHYHFNHLVRKGKKDRYGHNDNPSFKHHFVDSEGMVTEAGLLFFVSLFLEKRDAIWMQKKIRGFKGGTGPYEQMTNEVFCRSRISLPKLKLESLRTDDWMLLDMLNELVRCPKPLYDRLREKDRACFRVPVDILPDEDDTDGGGEDPFKNTLVRHQDRFPYFALRYFDLKKVFTSLRFHIDLGTYHFAIYKKMIGEQPEDRHLTRNLYGFGRIQDFAEEHRPEEWKRLVRDLDYFETGDKPYISQTTPHYHIEKGKIGLRFMPEGQHLWPSPEVGTTRTGRSKYAQDKRLTAEAFLSVHELMPMMFYYFLLREKYSEEVSAEKVQGRIKRVIKDVYAIYDAFARDEINTLKELDACSADKGIRRGHLPKQMIGILSQEHKNMEEKVRKKLQEMIADTDHRLDMLDRQTDRKIRIGRKNAGLPKSGVIADWLVRDMMRFQPVAKDTSGKPLNNSKANSTEYRMLQRALALFGGEKERLTPYFRQMNLTGGNNPHPFLDETRWESHTNILSFYRSYLRARKAFLERIGRSDRVENRPFLLLKEPKNDRQTLVAGWKSEFHLPRGIFTEAVRDCLIEMGYDEVGSYKEVGFMAKAVPLYFERACKDRVQPFYDSPFNVGNSLKPKKGRFLSKEKRAEEWESGKERFRLAKLKKEILEAKEHPYHDFKSWQKFERELRLVKNQDIITWMMCRDLMEENKVEGLDTGTLYLKDIRTDVHEQGSLNVLNRVKPMRLPVVVYRADSRGHVHKEQAPLATVYIEERDTKLLKQGNFKSFVKDRRLNGLFSFVDTGGLAMEQYPISKLRVEYELAKYQTARVCAFEQTLELEESLLTRYPHLPDENFREMLESWSDPLLGKWPDLHGKVRLLIAVRNAFSHNQYPMYDEAVFSSIRKYDPSSPDAIEERMGLNIAHRLSEEVKQAKETVERIIQA (SEQ ID NO: 97) PorphyromonasWP_ MTEQSERPYNGTYYTLEDKHFWAAFLNLARHNAYITLTHIDRQ gulae 039431778LAYSKADITNDQDVLSFKALWKNFDNDLERKSRLRSLILKHFSFLEGAAYGKKLFESKSSGNKSSKNKELTKKEKEELQANALSLDNLKSILFDFLQKLKDFRNYYSHYRHSESSELPLFDGNMLQRLYNVFDVSVQRVKRDHEHNDKVDPHRHFNHLVRKGKKDRYGHNDNPSFKHHFVDGEGMVTEAGLLFFVSLFLEKRDAIWMQKKIRGFKGGTETYQQMTNEVFCRSRISLPKLKLESLRTDDWMLLDMLNELVRCPKPLYDRLREDDRACFRVPVDILPDEDDTDGGGEDPFKNTLVRHQDRFPYFALRYFDLKKVFTSLRFHIDLGTYHFAIYKKMIGEQPEDRHLTRNLYGFGRIQDFAEEHRPEEWKRLVRDLDYFETGDKPYISQTSPHYHIEKGKIGLRFMPEGQHLWPSPEVGTTRTGRSKYAQDKRLTAEAFLSVHELMPMMFYYFLLREKYSEEVSAEKVQGRIKRVIEDVYAIYDAFARDEINTLKELDACLADKGIRRGHLPKQMIAILSQEHKDMEEKIRKKLQEMIADTDHRLDMLDRQTDRKIRIGRKNAGLPKSGVIADWLVRDMMRFQPVAKDTSGKPLNNSKANSTEYRMLQRALALFGGEKKRLTPYFRQMNLTGGNNPHPFLHETRWESHTNILSFYRSYLRARKAFLERIGRSDRMENRPFLLLKEPKTDRQTLVAGWKSEFHLPRGIFTEAVRDCLIEMGYDEVGSYREVGFMAKAVPLYFERACEDRVQPFYDSPFNVGNSLKPKKGRFLSKEERAEEWERGKERFRDLEAWSHSAARRIEDAFAGIEYASPGNKKKIEQLLRDLSLWEAFESKLKVRADKINLAKLKKEILEAQEHPYHDFKSWQKFERELRLVKNQDIITWMMCRDLMEENKVEGLDTGTLYLKDIRPNVQEQGSLNVLNRVKPMRLPVVVYRADSRGHVHKEEAPLATVYIEERDTKLLKQGNFKSFVKDRRLNGLFSFVDTGGLAMEQYPISKLRVEYELAKYQTARVCVFELTLRLEESLLTRYPHLPDESFRKMLESWSDPLLAKWPELHGKVRLLIAVRNAFSHNQYPMYDEAVESSIRKYDPSSPDAIEERMGLNIAHRLSEEVKQAKE TVERIIQV (SEQ ID NO: 98)Porphyromonas WP_ MTEQSERPYNGTYYTLEDKHFWAAFLNLARHNAYITLTHIDRQ gulae039437199 LAYSKADITNDEDILFFKGQWKNLDNDLERKSRLRSLILKHFSFLEGAAYGKKFFESKSSGNKSSKNKELTKKEKEELQANALSLDNLKSILFDFLQKLKDFRNYYSHYRHSGSSELPLFDGNMLQRLYNVFDVSVQRVKRDHEHNDEVDPHYHFNHLVRKGKKDRYGHNDNPSFKHHFVDGEGMVTEAGLLFFVSLFLEKRDAIWMQKKIRGFKGGTEPYEQMTNEVFCRSRISLPKLKLESLRTDDWMLLDMLNELVRCPKPLYDRLREKDRACFRVPVDILPDEDDTDGGGEDPFKNTLVRHQDRFPYFALRYFDLKKVFTSLRFHIDLGTYHFAIYKKMIGEQPEDRHLTRNLYGFGRIQDFAEEHRPEEWKRLVRDLDYFETGDKPYISQTTPHYHIEKGKIGLRFVPEGQHLWPSPEVGTTRTGRSKYAQDKRLTAEAFLSVHELMPMMFYYFLLREKYSEEVSAEKVQGRIKRVIEDVYAIYDAFARDEINTLKELDACLADKGIRRGHLPKQMIGILSQERKDMEEKVRKKLQEMIADTDHRLDMLDRQTDRKIRIGRKNAGLPKSGVIADWLVRDMMRFQPVAKDTSGKPLNNSKANSTEYRMLQRALALFGGEKERLTPYFRQMNLTGGNNPHPFLHETRWESHTNILSFYRSYLRARKAFLERIGRSDRVENCPFLLLKEPKTDRQTLVAGWKGEFHLPRGIFTEAVRDCLIEMGYDEVGSYREVGFMAKAVPLYFERACEDRVQPFYDSPFNVGNSLKPKKGRFLSKEKRAEEWESGKERFRLAKLKKEILEAQEHPYHDFKSWQKFERELRLVKNQDIITWMMCRDLMEENKVEGLDTGTLYLKDIRPNVQEQGSLNVLNRVKPMRLPVVVYRADSRGHVHKEEAPLATVYIEERDTKLLKQGNFKSFVKDRRLNGLFSFVDTGALAMEQYPISKLRVEYELAKYQTARVCAFEQTLELEESLLTRYPHLPDESFREMLESWSDPLLTKWPELHGKVRLLIAVRNAFSHNQYPMYDEAVESSIWKYDPSSPDAIEERMGLNIAHRLSEEVKQAKETIERIIQA (SEQ ID NO: 99) PorphyromonasWP_ MTEQSERPYNGTYYTLEDKHFWAAFLNLARHNAYITLTHIDRQ gulae 903442171LAYSKADITNDQDVLSFKALWKNLDNDLERKSRLRSLILKHFSFLEGAAYGKKLFESKSSGNKSSKNKELTKKEKEELQANALSLDNLKSILFDFLQKLKDFRNYYSHYRHSGSSELPLFDGNMLQRLYNVFDVSVQRVKRDHEHNDKVDPHYHFNHLVRKGKKDRYGHNDNPSFKHHFVDSEGMVTEAGLLFFVSLFLEKRDAIWMQKKIRGFKGGTGPYEQMTNEVFCRSRISLPKLKLESLRTDDWMLLDMLNELVRCPKPLYDRLREKDRACFRVPVDILPDEDDTDGGGEDPFKNTLVRHQDRFPYFALRYFDLKKVFTSLRFHIDLGTYHFAIYKKMIGEQPEDRHLTRNLYGFGRIQDFAEEHRPEEWKRLVRDLDYLETGDKPYISQTTPHYHIEKGKIGLRFVPEGQHLWPSPEVGTTRTGRSKCAQDKRLTAEAFLSVHELMPMMFYYFLLREKYSEEVSAEKVQGRIKRVIEDVYAIYDAFARDEINTLKELDTCLADKGIRRGHLPKQMITILSQERKDMKEKIRKKLQEMIADTDHRLDMLDRQTDRKIRIGRKNAGLPKSGVIADWLVRDMMRFQPVAKDASGKPLNNSKANSTEYRMLQRALALFGGEKERLTPYFRQMNLTGGNNPHPFLHETRWESHTNILSFYRSYLRARKAFLERIGRSDRVENCPFLLLKEPKTDRQTLVAGWKDEFHLPRGIFTEAVRDCLIEMGYDEVGSYREVGFMAKAVPLYFERACEDRVQPFYDSPFNVGNSLKPKKGRFLSKEDRAEEWERGMERFRDLEAWSHSAARRIKDAFAGIEYASPGNKKKIEQLLRDLSLWEAFESKLKVRADKINLAKLKKEILEAQEHPYHDFKSWQKFERELRLVKNQDIITWMMCRDLMEENKVEGLDTGTLYLKDIRPNVQEQGSLNVLNRVKPMRLPVVVYRADSRGHVHKEAPLATVYIEERNTKLLKQGNFKSFVKDRRLNGLFSFVDTGGLAMEQYPISKLRVEYELAKYQTARVCVFELTLRLEESLLSRYPHLPDESFREMLESWSDPLLAKWPELHGKVRLLIAVRNAFSHNQYPMYDEAVFSSIRKYDPSSPDAIEERMGLNIAHRLSEEVKQAKE TVERIIQA (SEQ ID NO: 100)Porphyromonas WP_ MNTVPATENKGQSRTVEDDPQYFGLYLNLARENLIEVESHVRI gulae039445055 KFGKKKLNEESLKQSLLCDHLLSIDRWTKVYGHSRRYLPFLHCFDPDSGIEKDHDSKTGVDPDSAQRLIRELYSLLDFLRNDFSHNRLDGTTFEHLKVSPDISSFITGAYTFACERAQSRFADFFKPDDELLAKNRKEQLISVADGKECLTVSGFAFFICLFLDREQASGMLSRIRGFKRTDENWARAVHETFCDLCIRHPHDRLESSNTKEALLLDMLNELNRCPRILYDMLPEEERAQFLPALDENSMNNLSENSLNEESRLLWDGSSDWAEALTKRIRHQDRFPYLMLRFIEEMDLLKGIRFRVDLGEIELDSYSKKVGRNGEYDRTITDHALAFGKLSDFQNEEEVSRMISGEASYPVRFSLFAPRYAIYDNKIGYCHTSDPVYPKSKTGEKRALSNPQSMGFISVHDLRKLLLMELLCEGSFSRMQSDFLRKANRILDETAEGKLQFSALFPEMRHRFIPPQNPKSKDRREKAETTLEKYKQEIKGRKDKLNSQLLSAFDMNQRQLPSRLLDEWMNIRPASHSVKLRTYVKQLNEDCRLRLRKFRKDGDGKARAIPLVGEMATFLSQDIVRMIISEETKKLITSAYYNEMQRSLAQYAGEENRRQFRAIVAELHLLDPSSGHPFLSATMETAHRYTEDFYKCYLEKKREWLAKTFYRPEQDENTKRRISVFFVPDGEARKLLPLLIRRRMKEQNDLQDWIRNKQAHPIDLPSHLFDSKIMELLKVKDGKKKWNEAFKDWWSTKYPDGMQPFYGLRRELNIHGKSVSYIPSDGKKFADCYTHLMEKTVRDKKRELRTAGKPVPPDLAAYIKRSFHRAVNEREFMLRLVQEDDRLMLMAINKMMTDREEDILPGLKNIDSILDEENQFSLAVHAKVLEKEGEGGDNSLSLVPATIEIKSKRKDWSKYIRYRYDRRVPGLMSHFPEHKATLDEVKTLLGEYDRCRIKIFDWAFALEGAIMSDRDLKPYLHESSSREGKSGEHSTLVKMLVEKKGCLTPDESQYLILIRNKAAHNQFPCAAEMPLIYRDVSAKVGSIEGSSAKDLPEGSSLVDSLWKKYEMIIRKILPILDHENRFFGKLLNNMSQPINDL (SEQ ID NO: 101) CapnocytoWP_ MENKTSLGNNIYYNPFKPQDKSYFAGYLNAAMENIDSVFRELG phaga 041989581KRLKGKEYTSENFFDAIFKENISLVEYERYVKLLSDYFPMARLL cynodegmiDKKEVPIKERKENFKKNFRGIIKAVRDLRNFYTHKEHGEVEITDEIFGVLDEMLKSTVLTVKKKKIKTDKTKEILKKSIEKQLDILCQKKLEYLKDTARKIEEKRRNQRERGEKKLVPRFEYSDRRDDLIAAIYNDAFDVYIDKKKDSLKESSKTKYNTESYPQQEEGDLKIPISKNGVVFLLSLFLSKQEVHAFKSKIAGFKATVIDEATVSHRKNSICFMATHEIFSHLAYKKLKRKVRTAEINYSEAENAEQLSIYAKETLMMQMLDELSKVPDVVYQNLSEDVQKTFIEDWNEYLKENNGDVGTMEEEQVIHPVIRKRYEDKFNYFAIRFLDEFAQFPTLRFQVHLGNYLHDSRPKEHLISDRRIKEKITVFGRLSELEHKKALFIKNTETNEDRKHYWEVFPNPNYDFPKENISVNDKDFPIAGSILDREKQPTAGKIGIKVNLLNQKYISEVDKAVKAHQLKQRNNKPSIQNIIEEIVPINGSNPKEIIVFGGQPTAYLSMNDIHSILYEFFDKWEKKKEKLEKKGEKELRKEIGKELEEKIVGKIQTQIQQIIDKDINAKILKPYQDDDSTAIDKEKLIKDLKQEQKILQKLKNEQTAREKEYQECIAYQEESRKIKRSDKSRQKYLRNQLKRKYPEVPTRKEILYYQEKGKVAVWLANDIKRFMPTDFKNEWKGEQHSLLQKSLAYYEQCKEELKNLLPQQKVFKHLPFELGGHFQQKYLYQFYTRYLDKRLEHISGLVQQAENFKNENKVFKKVENECFKFLKKQNYTHKGLDAQAQSVLGYPIFLERGFMDEKPTIIKGKTFKGNESLFTDWFRYYKEYQNFQTFYDTENYPLVELEKKQADRKRETKIYQQKKNDVFTLLMAKHIFKSVFKQDSIDRFSLEDLYQSREERLENQEKAKQTGERNTNYIWNKTVDLNLCDGKVTVENVKLKNVGNFIKYEYDQRVQTFLKYEENIKWQAFLIKESKEEENYPYIVEREIEQYEKVRREELLKEVHLIEEYILEKVKDKEILKKGDNQNFKYYILNGLLKQLKNEDVESYKVFNLNTKPEDVNINQLKQEATDLEQKAFVLTYIRNKFAHNQLPKKEFWDYCQEKYGKIEKEKTYAEYFAEVFKREKEALMK (SEQ ID NO: 102) Prevotella WP_MNIPALVENQKKYFGTYSVMAMLNAQTVLDHIQKVADIEGEQ sp. P5-119 042518169NENNENLWFHPVMSHLYNAKNGYDKQPEKTMFIIERLQSYFPFLKIMAENQREYSNGKYKQNRVEVNSNDIFEVLKRAFGVLKMYRDLTNHYKTYEEKLIDGCEFLTSTEQPLSGMISKYYTVALRNTKERYGYKTEDLAFIQDNIKKITKDAYGKRKSQVNTGFFLSLQDYNGDTQKKLHLSGVGIALLICLFLDKQYINIFLSRLPIFSSYNAQSEERRIIIRSFGINSIKLPKDRIHSEKSNKSVAMDMLNEVKRCPDELFTTLSAEKQSRFRIISDDHNEVLMKRSTDRFVPLLLQYIDYGKLFDHIRFHVNMGKLRYLLKADKTCIDGQTRVRVIEQPLNGFGRLEEAETMRKQENGTFGNSGIRIRDFENVKRDDANPANYPYIVDTYTHYILENNKVEMFISDKGSSAPLLPLIEDDRYVVKTIPSCRMSTLEIPAMAFHMFLFGSKKTEKLIVDVHNRYKRLFQAMQKEEVTAENIASFGIAESDLPQKILDLISGNAHGKDVDAFIRLTVDDMLTDTERRIKRFKDDRKSIRSADNKMGKRGFKQISTGKLADFLAKDIVLFQPSVNDGENKITGLNYRIMQSAIAVYDSGDDYEAKQQFKLMFEKARLIGKGTTEPHPFLYKVFARSIPANAVDFYERYLIERKFYLTGLCNEIKRGNRVDVPFIRRDQNKWKTPAMKTLGRIYSEDLPVELPRQMFDNEIKSHLKSLPQMEGIDENNANVTYLIAEYMKRVLNDDFQTFYQWKRNYHYMDMLKGEYDRKGSLQHCFTSVEEREGLWKERASRTERYRKLASNKIRSNRQMRNASSEEIETILDKRLSNCRNEYQKSEKVIRRYRVQDALLFLLAKKTLTELADFDGERFKLKEIMPDAEKGILSEIMPMSFTFEKGGKKYTITSEGMKLKNYGDFFVLASDKRIGNLLELVGSDIVSKEDIMEEFNKYDQCRPEISSIVENLEKWAFDTYPELSARVDREEKVDFKSILKILLNNKNINKEQSDILRKIRNAFDHNNYPDKGIVEIKALPEIAMSIKKAFGEYAIMK (SEQ ID NO: 103) Prevotella WP_MNIPALVENQKKYFGTYSVMAMLNAQTVLDHIQKVADIEGEQ sp. P4-76 044072147NENNENLWFHPVMSHLYNAKNGYDKQPEKTMFIIERLQSYFPFLKIMAENQREYSNGKYKQNRVEVNSNDIFEVLKRAFGVLKMYRDQASHYKTYDEKLIDGCEFLTSTEQPLSGMINNYYTVALRNMNERYGYKTEDLAFIQDKRFKFVKDAYGKKKSQVNTGFFLSLQDYNGDTQKKLHLSGVGIALLICLFLDKQYINIFLSRLPIFSSYNAQSEERRIIIRSFGINSIKQPKDRIHSEKSNKSVAMDMLNEIKRCPNELFETLSAEKQSRFRIISNDHNEVLMKRSSDRFVPLLLQYIDYGKLFDHIRFHVNMGKLRYLLKADKTCIDGQTRVRVIEQPLNGFGRLEEVETMRKQENGTFGNSGIRIRDFENMKRDDANPANYPYIVDTYTHYILENNKVEMFISDEETPAPLLPVIEDDRYVVKTIPSCRMSTLEIPAMAFHMFLFGSKKTEKLIVDVHNRYKRLFKAMQKEEVTAENIASFGIAESDLPQKIIDLISGNAHGKDVDAFIRLTVDDMLADTERRIKRFKDDRKSIRSADNKMGKRGFKQISTGKLADFLAKDIVLFQPSVNDGENKITGLNYRIMQSAIAVYNSGDDYEAKQQFKLMFEKARLIGKGTTEPHPFLYKVFVRSIPANAVDFYERYLIERKFYLIGLSNEIKKGNRVDVPFIRRDQNKWKTPAMKTLGRIYDEDLPVELPRQMFDNEIKSHLKSLPQMEGIDENNANVTYLIAEYMKRVLNDDFQTFYQWKRNYRYMDMLRGEYDRKGSLQSCFTSVEEREGLWKERASRTERYRKLASNKIRSNRQMRNASSEEIETILDKRLSNSRNEYQKSEKVIRRYRVQDALLFLLAKKTLTELADFDGERFKLKEIMPDAEKGILSEIMPMSFTFEKGGKKYTITSEGMKLKNYGDFFVLASDKRIGNLLELVGSDTVSKEDIMEEFKKYDQCRPEISSIVFNLEKWAFDTYPELSARVDREEKVDFKSILKILLNNKNINKEQSDILRKIRNAFDHNNYPDKGVVEIRALPEIAMSIKKAFGEYAIMK (SEQ ID NO: 104) Prevotella WP_MNIPALVENQKKYFGTYSVMAMLNAQTVLDHIQKVADIEGEQ sp. P5-60 044074780NENNENLWFHPVMSHLYNAKNGYDKQPEKTMFIIERLQSYFPFLKIMAENQREYSNGKYKQNRVEVNSNDIFEVLKRAFGVLKMYRDLTNHYKTYEEKLIDGCEFLTSTEQPFSGMISKYYTVALRNTKERYGYKAEDLAFIQDNRYKFTKDAYGKRKSQVNTGSFLSLQDYNGDTTKKLHLSGVGIALLICLFLDKQYINLFLSRLPIFSSYNAQSEERRIIIRSFGINSIKQPKDRIHSEKSNKSVAMDMLNEVKRCPDELFTTLSAEKQSRFRIISDDHNEVLMKRSSDRFVPLLLQYIDYGKLFDHIRFHVNMGKLRYLLKADKTCIDGQTRVRVIEQPLNGFGRLEEVETMRKQENGTFGNSGIRIRDFENMKRDDANPANYPYIVETYTHYILENNKVEMFISDEENPTPLLPVIEDDRYVVKTIPSCRMSTLEIPAMAFHMFLFGSEKTEKLIIDVHDRYKRLFQAMQKEEVTAENIASFGIAESDLPQKIMDLISGNAHGKDVDAFIRLTVDDMLTDTERRIKRFKDDRKSIRSADNKMGKRGFKQISTGKLADFLAKDIVLFQPSVNDGENKITGLNYRIMQSAIAVYDSGDDYEAKQQFKLMFEKARLIGKGTTEPHPFLYKVFVRSIPANAVDFYERYLIERKFYLIGLSNEIKKGNRVDVPFIRRDQNKWKTPAMKTLGRIYSEDLPVELPRQMFDNEIKSHLKSLPQMEGIDENNANVTYLIAEYMKRVLNDDFQTFYQWKRNYRYMDMLRGEYDRKGSLQHCFTSIEEREGLWKERASRTERYRKLASNKIRSNRQMRNASSEEIETILDKRLSNCRNEYQKSEKIIRRYRVQDALLFLLAKKTLTELADFDGERFKLKEIMPDAEKGILSEIMPMSFTFEKGGKIYTITSGGMKLKNYGDFFVLASDKRIGNLLELVGSNTVSKEDIMEEFKKYDQCRPEISSIVFNLEKWAFDTYPELPARVDRKEKVDFWSILDVLSNNKDINNEQSYILRKIRNAFDHNNYPDKGIVEIKALPEIAMSIKKAFGEYAIMK (SEQ ID NO: 105) Phaeodactyli-WP_ MTNTPKRRTLHRHPSYFGAFLNIARHNAFMIMEHLSTKYDMED bacter 044218239KNTLDEAQLPNAKLFGCLKKRYGKPDVTEGVSRDLRRYFPFLN xiamenensisYPLFLHLEKQQNAEQAATYDINPEDIEFTLKGFFRLLNQMRNNYSHYISNTDYGKFDKLPVQDIYEAAIFRLLDRGKHTKRFDVFESKHTRHLESNNSEYRPRSLANSPDHENTVAFVTCLFLERKYAFPFLSRLDCFRSTNDAAEGDPLIRKASHECYTMFCCRLPQPKLESSDILLDMVNELGRCPSALYNLLSEEDQARFHIKREEITGFEEDPDEELEQEIVLKRHSDRFPYFALRYFDDTEAFQTLRFDVYLGRWRTKPVYKKRIYGQERDRVLTQSIRTFTRLSRLLPIYENVKHDAVRQNEEDGKLVNPDVTSQFHKSWIQIESDDRAFLSDRIEHFSPHYNFGDQVIGLKFINPDRYAAIQNVFPKLPGEEKKDKDAKLVNETADAIISTHEIRSLFLYHYLSKKPISAGDERRFIQVDTETFIKQYIDTIKLFFEDIKSGELQPIADPPNYQKNEPLPYVRGDKEKTQEERAQYRERQKEIKERRKELNTLLQNRYGLSIQYIPSRLREYLLGYKKVPYEKLALQKLRAQRKEVKKRIKDIEKMRTPRVGEQATWLAEDIVFLTPPKMHTPERKTTKHPQKLNNDQFRIMQSSLAYFSVNKKAIKKFFQKETGIGLSNRETSHPFLYRIDVGRCRGILDFYTGYLKYKMDWLDDAIKKVDNRKHGKKEAKKYEKYLPSSIQHKTPLELDYTRLPVYLPRGLFKKAIVKALAAHADFQVEPEEDNVIFCLDQLLDGDTQDFYNWQRYYRSALTEKETDNQLVLAHPYAEQILGTIKTLEGKQKNNKLGNKAKQKIKDELIDLKRAKRRLLDREQYLRAVQAEDRALWLMIQERQKQKAEHEEIAFDQLDLKNITKILTESIDARLRIPDTKVDITDKLPLRRYGDLRRVAKDRRLVNLASYYHVAGLSEIPYDLVKKELEEYDRRRVAFFEHVYQFEKEVYDRYAAELRNENPKGESTYFSHWEYVAVAVKHSADTHFNELFKEKVMQLRNKFHHNEFPYFDWLLPEVEKASAALYADRVFDVAEGYYQKMRKLMRQ (SEQ ID NO: 106) FlavobacteriumWP_ MDNNITVEKTELGLGITYNHDKVEDKHYFGGFFNLAQNNIDLV sp. 316 045968377AQEFKKRLLIQGKDSINIFANYFSDQCSITNLERGIKILAEYFPVVSYIDLDEKNKSKSIREHLILLLETINNLRNYYTHYYHKKIIIDGSLFPLLDTILLKVVLEIKKKKLKEDKTKQLLKKGLEKEMTILFNLMKAEQKEKKIKGWNIDENIKGAVLNRAFSHLLYNDELSDYRKSKYNTEDETLKDTLTESGILFLLSFFLNKKEQEQLKANIKGYKGKIASIPDEEITLKNNSLRNMATHWTYSHLTYKGLKHRIKTDHEKETLLVNMVDYLSKVPHEIYQNLSEQNKSLFLEDINEYMRDNEENHDSSEASRVIHPVIRKRYENKFAYFAIRFLDEFAEFPTLRFMVNVGNYIHDNRKKDIGGTSLITNRTIKQQINVFGNLTEIHKKKNDYFEKEENKEKTLEWELFPNPSYHFQKENIPIFIDLEKSKETNDLAKEYAKEKKKIFGSSRKKQQNTAKKNRETIINLVFDKYKTSDRKTVTFEQPTALLSFNELNSFLYAFLVENKTGKELEKIIIEKIANQYQILKNCSSTVDKTNDNIPKSIKKIVNTTTDSFYFEGKKIDIEKLEKDITIEIEKTNEKLETIKENEESAQNYKRNERNTQKRKLYRKYVFFTNEIGIEATWITNDILRFLDNKENWKGYQHSELQKFISQYDNYKKEALGLLESEWNLESDAFFGQNLKRMFQSNSTFETFYKKYLDNRKNTLETYLSAIENLKTMTDVRPKVLKKKWTELFRFFDKKIYLLSTIETKINELITKPINLSRGIFEEKPTFINGKNPNKENNQHLFANWFIYAKKQTILQDFYNLPLEQPKAITNLKKHKYKLERSINNLKIEDIYIKQMVDFLYQKLFEQSFIGSLQDLYTSKEKREIEKGKAKNEQTPDESFIWKKQVEINTHNGRIIAKTKIKDIGKFKNLLTDNKIAHLISYDDRIWDFSLNNDGDITKKLYSINTELESYETIRREKLLKQIQQFEQFLLEQETEYSAERKHPEKFEKDCNPNFKKYIIEGVLNKIIPNHEIEEIEILKSKEDVFKINFSDILILNNDNIKKGYLLIMIRNKFAHNQLIDKNLFNFSLQLYSKNENENFSEYLNKVCQNIIQEFKEKLK (SEQ ID NO: 107) Porphyromonas WP_MTEQSERPYNGTYYTLEDKHFWAAFLNLARHNAYITLTHIDRQ gulae 604201018LAYSKADITNDQDVLSFKALWKNFDNDLERKSRLRSLILKHFSFLEGAAYGKKLFESKSSGNKSSKNKELTKKEKEELQANALSLDNLKSILFDFLQKLKDFRNYYSHYRHSESSELPLFDGNMLQRLYNVFDVSVQRVKRDHEHNDKVDPHRHFNHLVRKGKKDRYGHNDNPSFKHHFVDSEGMVTEAGLLFFVSLFLEKRDAIWMQKKIRGFKGGTETYQQMTNEVFCRSRISLPKLKLESLRTDDWMLLDMLNELVRCPKPLYDRLREKDRARFRVPVDILPDEDDTDGGGEDPFKNTLVRHQDRFPYFALRYFDLKKVFTSLRFHIDLGTYHFAIYKKMIGEQPEDRHLTRNLYGFGRIQDFAEEHRPEEWKRLVRDLDYFETGDKPYISQTTPHYHIEKGKIGLRFMPEGQHLWPSPEVGTTRTGRSKYAQDKRLTAEAFLSVHELMPMMFYYFLLREKYSEEVSAEKVQGRIKRVIEDVYAIYDAFARDEINTLKELDACLADKGIRRGHLPKQMIAILSQEHKDMEEKIRKKLQEMIADTDHRLDMLDRQTDRKIRIGRKNAGLPKSGVIADWLVRDMMRFQPVAKDTSGKPLNNSKANSTEYRMLQRALALFGGEKKRLTPYFRQMNLTGGNNPHPFLHETRWESHTNILSFYRSYLRARKAFLERIGRSDRMENRPFLLLKEPKTDRQTLVAGWKSEFHLPRGIFTEAVRDCLIEMGYDEVGSYREVGFMAKAVPLYFERACEDRVQPFYDSPFNVGNSLKPKKGRFLSKEERAEEWERGKERFRDLEAWSHSAARRIEDAFAGIEYASPGNKKKIEQLLRDLSLWEAFESKLKVRADKINLAKLKKEILEAQEHPYHDFKSWQKFERELRLVKNQDIITWMMCRDLMEENKVEGLDTGTLYLKDIRPNVQEQGSLNVLNRVKPMRLPVVVYRADSRGHVHKEEAPLATVYIEERDTKLLKQGNFKSFVKDRRLNGLFSFVDTGGLAMEQYPISKLRVEYELAKYQTARVCVFELTLRLEESLLTRYPHLPDESFRKMLESWSDPLLAKWPELHGKVRLLIAVRNAFSHNQYPMYDEAVESSIRKYDPSSPDAIEERMGLNIAHRLSEEVKQAKE TVERIIQV (SEQ ID NO: 108)WP_04743 Chryseo METQTIGHGIAYDHSKIQDKHFFGGFLNLAENNIKAVLKAFSEK 1796bacterium FNVGNVDVKQFADVSLKDNLPDNDFQKRVSFLKMYFPVVDFIN sp.IPNNRAKFRSDLTTLFKSVDQLRNFYTHYYHKPLDFDASLFILLD YR477DIFARTAKEVRDQKMKDDKTRQLLSKSLSEELQKGYELQLERLKELNRLGKKVNIHDQLGIKNGVLNNAFNHLIYKDGESFKTKLTYSSALTSFESAENGIEISQSGLLFLLSMFLKRKEIEDLKNRNKGFKAKVVIDEDGKVNGLKFMATHWVFSYLCFKGLKSKLSTEFHEETLLIQIIDELSKVPDELYCAFDKETRDKFIEDINEYVKEGHQDFSLEDAKVIHPVIRKRYENKFNYFAIRFLDEFVKFPSLRFQVHVGNYVHDRRIKNIDGTTFETERVVKDRIKVFGRLSEISSYKAQYLSSVSDKHDETGWEIFPNPSYVFINNNIPIHISVDTSFKKEIADFKKLRRAQVPDELKIRGAEKKRKFEITQMIGSKSVLNQEEPIALLSLNEIPALLYEILINGKEPAEIERIIKDKLNERQDVIKNYNPENWLPASQISRRLRSNKGERIINTDKLLQLVTKELLVTEQKLKIISDNREALKQKKEGKYIRKFIFTNSELGREAIWLADDIKRFMPADVRKEWKGYQHSQLQQSLAFYNSRPKEALAILESSWNLKDEKIIWNEWILKSFTQNKFFDAFYNEYLKGRKKYFAFLSEHIVQYTSNAKNLQKFIKQQMPKDLFEKRHYIIEDLQTEKNKILSKPFIFPRGIFDKKPTFIKGVKVEDSPESFANWYQYGYQKDHQFQKFYDWKRDYSDVFLEHLGKPFINNGDRRTLGMEELKERIIIKQDLKIKKIKIQDLFLRLIAENLFQKVFKYSAKLPLSDFYLTQEERMEKENMAALQNVREEGDKSPNIIKDNFIWSKMIPYKKGQIIENAVKLKDIGKLNVLSLDDKVQTLLSYDDAKPWSKIALENEFSIGENSYEVIRREKLFKEIQQFESEILFRSGWDGINHPAQLEDNRNPKFKMYIVNGILRKSAGLYSQGEDIWFEYNADFNNLDADVLETKSELVQLAFLVTAIRNKFAHNQLPAKEFYFYIRAKYGFADEPSVALVYLNFTKYAINEFKKVMI (SEQ ID NO: 109) Riemerella WP_MFFSFHNAQRVIFKHLYKAFDASLRMVKEDYKAHFTVNLTRDF anatipestifer 904354263AHLNRKGKNKQDNPDFNRYRFEKDGFFTESGLLFFTNLFLDKRDAYWMLKKVSGFKASHKQREKMTTEVFCRSRILLPKLRLESRYDHNQMLLDMLSELSRCPKLLYEKLSEENKKHFQVEADGFLDEIEEEQNPFKDTLIRHQDRFPYFALRYLDLNESFKSIRFQVDLGTYHYCIYDKKIGDEQEKRHLTRTLLSFGRLQDFTEINRPQEWKALTKDLDYKETSNQPFISKTTPHYHITDNKIGFRLGTSKELYPSLEIKDGANRIAKYPYNSGFVAHAFISVHELLPLMFYQHLTGKSEDLLKETVRHIQRIYKDFEEERINTIEDLEKANQGRLPLGAFPKQMLGLLQNKQPDLSEKAKIKIEKLIAETKLLSHRLNTKLKSSPKLGKRREKLIKTGVLADWLVKDFMRFQPVAYDAQNQPIKSSKANSTEFWFIRRALALYGGEKNRLEGYFKQTNLIGNTNPHPFLNKFNWKACRNLVDFYQQYLEQREKFLEAIKNQPWEPYQYCLLLKIPKENRKNLVKGWEQGGISLPRGLFTEAIRETLSEDLMLSKPIRKEIKKHGRVGFISRAITLYFKEKYQDKHQSFYNLSYKLEAKAPLLKREEHYEYWQQNKPQSPTESQRLELHTSDRWKDYLLYKRWQHLEKKLRLYRNQDVMLWLMTLELTKNHFKELNLNYHQLKLENLAVNVQEADAKLNPLNQTLPMVLPVKVYPATAFGEVQYHKTPIRTVYIREEHTKALKMGNFKALVKDRRLNGLFSFIKEENDTQKHPISQLRLRRELEIYQSLRVDAFKETLSLEEKLLNKHTSLSSLENEFRALLEEWKKEYAASSMVTDEHIAFIASVRNAFCHNQYPFYKEALHAPIPLFTVAQPTTEEKDGLGIAEALLKVLREYCEIVKSQI (SEQ ID NO: 110) Porphyromonas WP_MTEQNEKPYNGTYYTLEDKHFWAAFFNLARHNAYITLAHIDRQ gingivalis 052912312LAYSKADITNDEDILFFKGQWKNLDNDLERKARLRSLILKHFSFLEGAAYGKKLFESQSSGNKSSKKKELTKKEKEELQANALSLDNLKSILFDFLQKLKDFRNYYSHYRHPESSELPLFDGNMLQRLYNVFDVSVQRVKRDHEHNDKVDPHRHFNHLVRKGKKDKYGNNDNPFFKHHFVDREEKVTEAGLLFFVSLFLEKRDAIWMQKKIRGFKGGTEAYQQMTNEVFCRSRISLPKLKLESLRTDDWMLLDMLNELVRCPKLLYDRLREEDRARFRVPVDILSDEDDTDGTEEDPFKNTLVRHQDRFPYFALRYFDLKKVFTSLRFHIDLGTYHFAIYKKNIGEQPEDRHLTRNLYGFGRIQDFAEEHRPEEWKRLVRDLDYFETGDKPYITQTTPHYHIEKGKIGLRFVPEGQLLWPSPEVGATRTGRSKYAQDKRFTAEAFLSVHELMPMMFYYFLLREKYSEEASAEKVQGRIKRVIEDVYAVYDAFARDEINTRDELDACLADKGIRRGHLPRQMIAILSQEHKDMEEKVRKKLQEMIADTDHRLDMLDRQTDRKIRIGRKNAGLPKSGVIADWLVRDMMRFQPVAKDTSGKPLNNSKANSTEYRMLQRALALFGGEKERLTPYFRQMNLTGGNNPHPFLHETRWESHTNILSFYRSYLKARKAFLQSIGRSDREENHRFLLLKEPKTDRQTLVAGWKSEFHLPRGIFTEAVRDCLIEMGYDEVGSYKEVGFMAKAVPLYFERACKDRVQPFYDYPFNVGNSLKPKKGRFLSKEKRAEEWESGKERFRDLEAWSHSAARRIEDAFVGIEYASWENKKKIEQLLQDLSLWETFESKLKVKADKINIAKLKKEILEAKEHPYHDFKSWQKFERELRLVKNQDIITWMMCRDLMEENKVEGLDTGTLYLKDIRTDVQEQGSLNVLNHVKPMRLPVVVYRADSRGHVHKEEAPLATVYIEERDTKLLKQGNFKSFVKDRRLNGLFSFVDTGALAMEQYPISKLRVEYELAKYQTARVCAFEQTLELEESLLTRYPHLPDESFREMLESWSDPLLDKWPDLQREVRLLIAVRNAFSHNQYPMYDETIFSSIRKYDPSSLDAIEERMGLNIAHRLSEEVKLAKEMV ERIIQA (SEQ ID NO: 111)Porphyromonas WP_ MTEQNEKPYNGTYYTLKDKHFWAAFFNLARHNAYITLTHIDRQ gingivalis058019250 LAYSKADITNDEDILFFKGQWKNLDNDLERKARLRSLILKHFSFLEGAAYGKKLFESQSSGNKSSKKKELTKKEKEELQANALSLDNLKSILFDFLQKLKDFRNYYSHYRHPESSELPMFDGNMLQRLYNVFDVSVQRVKRDHEHNDKVDPHRHFNHLVRKGKKDRCGNNDNPFFKHHFVDREGKVTEAGLLFFVSLFLEKRDAIWMQKKIRGFKGGTETYQQMTNEVFCRSRISLPKLKLESLRTDDWMLLDMLNELVRCPKSLYDRLREEDRACFRVPVDILSDEDDTDGAEEDPFKNTLVRHQDRFPYFALRYFDLKKVFTSLRFHIDLGTYHFAIYKKNIGEQPEDRHLTRNLYGFGRIQDFAEEHRPEEWKRLVRDLDCFETGDKPYITQTTPHYHIEKGKIGLRFVPEGQHLWPSPEVGATRTGRSKYAQDKRFTAEAFLSVHELMPMMFYYFLLREKYSEEVSAERVQGRIKRVIEDVYAVYDAFARDEINTRDELDACLADKGIRRGHLPRQMIAILSQKHKDMEEKVRKKLQEMIADTDHRLDMLDRQTDRKIRIGRKNAGLPKSGVIADWLVRDMMRFQPVAKDTSGKPLNNSKANSTEYRMLQRALALFGGEKERLTPYFRQMNLTGGNNPHPFLHETRWESHTNILSFYRSYLKARKAFLQSIGRSDRVENHRFLLLKEPKTDRQTLVAGWKGEFHLPRGIFTEAVRDCLIEMGLDEVGSYKEVGFMAKAVPLYFERACKDRVQPFYDYPFNVGNSLKPKKGRFLSKEKRAEEWESGKERFRDLEAWSHSAARRIEDAFAGIENASRENKKKIEQLLQDLSLWETFESKLKVKADKINIAKLKKEILEAKEHPYLDFKSWQKFERELRLVKNQDIITWMMCRDLMEENKVEGLDTGTLYLKDIRTDVQEQGSLNVLNHVKPMRLPVVVYRADSRGHVHKEQAPLATVYIEERDTKLLKQGNFKSFVKDRRLNGLFSFVDTGALAMEQYPISKLRVEYELAKYQTARVCAFEQTLELEESLLTRYPHLPDENFRKMLESWSDPLLDKWPDLHRKVRLLIAVRNAFSHNQYPMYDEAVFSSIRKYDPSSPDAIEERMGLNIAHRLSEEVKQAKEMAERIIQA (SEQ ID NO: 112) Flavobacterium WP_MSSKNESYNKQKTFNHYKQEDKYFFGGFLNNADDNLRQVGKE columnare 060381855FKTRINFNHNNNELASVFKDYFNKEKSVAKREHALNLLSNYFPVLERIQKHTNHNFEQTREIFELLLDTIKKLRDYYTHHYHKPITINPKVYDFLDDTLLDVLITIKKKKVKNDTSRELLKEKFRPELTQLKNQKREELIKKGKKLLEENLENAVFNHCLRPFLEENKTDDKQNKTVSLRKYRKSKPNEETSITLTQSGLVFLISFFLHRKEFQVFTSGLEGFKAKVNTIKEEEISLNKNNIVYMITHWSYSYYNFKGLKHRIKTDQGVSTLEQNNTTHSLTNTNTKEALLTQIVDYLSKVPNEIYETLSEKQQKEFEEDINEYMRENPENEDSTFSSIVSHKVIRKRYENKFNYFAMRFLDEYAELPTLRFMVNFGDYIKDRQKKILESIQFDSERIIKKEIHLFEKLGLVTEYKKNVYLKETSNIDLSRFPLFPSPSYVMANNNIPFYIDSRSNNLDEYLNQKKKAQSQNRKRNLTFEKYNKEQSKDAIIAMLQKEIGVKDLQQRSTIGLLSCNELPSMLYEVIVKDIKGAELENKIAQKIREQYQSIRDFTLDSPQKDNIPTTLTKTISTDTSVTFENQPIDIPRLKNALQKELTLTQEKLLNVKQHEIEVDNYNRNKNTYKFKNQPKDKVDDNKLQRKYVFYRNEIGQEANWLASDLIHFMKNKSLWKGYMHNELQSFLAFFEDKKNDCIALLETVFNLKEDCILTKDLKNLFLKHGNFIDFYKEYLKLKEDFLNTESTFLENGFIGLPPKILKKELSKRLNYIFIVFQKRQFIIKELEEKKNNLYADAINLSRGIFDEKPTMIPFKKPNPDEFASWFVASYQYNNYQSFYELTPDKIENDKKKKYKNLRAINKVKIQDYYLKLMVDTLYQDLFNQPLDKSLSDFYVSKTDREKIKADAKAYQKRNDSFLWNKVIHLSLQNNRITANPKLKDIGKYKRALQDEKIATLLTYDDRTWTYALQKPEKENENDYKELHYTALNMELQEYEKVRSKKLLKQVQELEKQILDKFYDFSNNATHPEDLEIEDKKGKRHPNFKLYITKALLKNESEIINLENIDIEILIKYYDYNTEKLKEKIKNMDEDEKAKIVNTKENYNKITNVLIKKALVLIIIRNKMAHNQYPPKFIYDLATRFVPKKEEEYFACYFNRVFETITTELWENKKKAKEIV (SEQ ID NO: 113) Porphyromonas WP_MTEQNERPYNGTYYTLEDKHFWAAFFNLARHNAYITLTHIDRQ gingivalis 061156470LAYSKADITNDEDILFFKGQWKNLDNDLERKARLRSLILKHFSFLEGAAYGKKLFENKSSGNKSSKKKELTKKEKEELQANALSLDNLKSILFDFLQKLKDFRNYYSHYRHPESSELPLFDGNMLQRLYNVFDVSVQRVKRDHEHNDKVDPHRHFNHLVRKGKKDRCGNNDNPFFKHHFVDREGKVTEAGLLFFVSLFLEKRDAIWMQKKIRGFKGGTEAYQQMTNEVFCRSRISLPKLKLESLRTDDWMLLDMLNELVRCPKSLYDRLREEDRARFRVPVDILSDEDDTDGTEEDPFKNTLVRHQDRFPYFALRYFDLKKVFTSLRFHIDLGTYHFAIYKKNIGEQPEDRHLTRNLYGFGRIQDFAEEHRPEEWKRLVRDLDYFETGDKPYITQTTPHYHIEKGKIGLRFVPEGQHLWPSPEVGATRTGRSKYAQDKRLTAEAFLSVHELMPMMFYYFLLREKYSEEVSAEKVQGRIKRVIEDVYAVYDAFARGEIDTLDRLDACLADKGIRRGHLPRQMIAILSQEHKDMEEKVRKKLQEMIADTDHRLDMLDRQTDRKIRIGRKNAGLPKSGVIADWLVRDMMRFQPVAKDTSGKPLNNSKANSTEYRMLQRALALFGGEKERLTPYFRQMNLTGGNNPHPFLHETRWESHTNILSFYRSYLKARKAFLQSIGRSDREENHRFLLLKEPKTDRQTLVAGWKSEFHLPRGIFTEAVRDCLIEMGYDEVGSYKEVGFMAKAVPLYFERACKDRVQPFYDYPFNVGNSLKPKKGRFLSKEKRAEEWESGKERFRLAKLKKEILEAKEHPYLDFKSWQKFERELRLVKNQDIITWMMCRDLMEENKVEGLDTGTLYLKDIRTEVQEQGSLNVLNRVKPMRLPVVVYRADSRGHVHKEQAPLATVYIEERDTKLLKQGNFKSFVKDRRLNGLFSFVDTGGLAMEQYPISKLRVEYELAKYQTARVCAFEQTLELEESLLTRCPHLPDKNFRKMLESWSDPLLDKWPDLQREVWLLIAVRNAFSHNQYPMYDEAVFSSIRKYDPSSPDAIEERMGLNIAHRLSEEVKQAKEMAERIIQA (SEQ ID NO: 114) PorphyromonasWP_ MNTVPASENKGQSRTVEDDPQYFGLYLNLARENLIEVESHVRIK gingivalis 061156637FGKKKLNEESLKQSLLCDHLLSVDRWTKVYGHSRRYLPFLHYFDPDSQIEKDHDSKTGVDPDSAQRLIRELYSLLDFLRNDFSHNRLDGTTFEHLEVSPDISSFITGTYSLACGRAQSRFADFFKPDDFVLAKNRKEQLISVADGKECLTVSGLAFFICLFLDREQASGMLSRIRGFKRTDENWARAVHETFCDLCIRHPHDRLESSNTKEALLLDMLNELNRCPRILYDMLPEEERAQFLPALDENSMNNLSENSLNEESRLLWDGSSDWAEALTKRIRHQDRFPYLMLRFIEEMDLLKGIRFRVDLGEIELDSYSKKVGRNGEYDRTITDHALAFGKLSDFQNEEEVSRMISGEASYPVRFSLFAPRYAIYDNKIGYCHTSDPVYPKSKTGEKRALSNPQSMGFISVHDLRKLLLMELLCEGSFSRMQSGFLRKANRILDETAEGKLQFSALFPEMRHRFIPPQNPKSKDRREKAETTLEKYKQEIKGRKDKLNSQLLSAFDMNQRQLPSRLLDEWMNIRPASHSVKLRTYVKQLNEDCRLRLRKFRKDGDGKARAIPLVGEMATFLSQDIVRMIISEETKKLITSAYYNEMQRSLAQYAGEENRRQFRAIVAELHLLDPSSGHPFLSATMETAHRYTEDFYKCYLEKKREWLAKTFYRPEQDENTKRRISVFFVPDGEARKLLPLLIRRRMKEQNDLQDWIRNKQAHPIDLPSHLFDSKIMELLKVKDGKKKWNEAFKDWWSTKYPDGMQPFYGLRRELNIHGKSVSYIPSDGKKFADCYTHLMEKTVQDKKRELRTAGKPVPPDLAADIKRSFHRAVNEREFMLRLVQEDDRLMLMAINKMMTDREEDILPGLKNIDSILDKENQFSLAVHAKVLEKEGEGGDNSLSLVPATIEIKSKRKDWSKYIRYRYDRRVPGLMSHFPEHKATLDEVKTLLGEYDRCRIKIFDWAFALEGAIMSDRDLKPYLHESSSREGKSGEHSTLVKMLVEKKGCLTPDESQYLILIRNKAAHNQFPCAAEMPLIYRDVSAKVGSIEGSSAKDLPEGSSLVDSLWKKYEMIIRKILPILDPENRFFGKLLNNMSQPINDL (SEQ ID NO: 115) RiemerellaWP_ MFFSFHNAQRVIFKHLYKAFDASLRMVKEDYKAHFTVNLTRDF anatipestifer 061710138AHLNRKGKNKQDNPDFNRYRFEKDGFFTESGLLFFTNLFLDKRDAYWMLKKVSGFKASHKQSEKMTTEVFCRSRILLPKLRLESRYDHNQMLLDMLSELSRCPKLLYEKLSEKDKKCFQVEADGFLDEIEEEQNPFKDTLIRHQDRFPYFALRYLDLNESFKSIRFQVDLGTYHYCIYDKKIGYEQEKRHLTRTLLNFGRLQDFTEINRPQEWKALTKDLDYNETSNQPFISKTTPHYHITDNKIGFRLRTSKELYPSLEVKDGANRIAKYPYNSDFVAHAFISISVHELLPLMFYQHLTGKSEDLLKETVRHIQRIYKDFEEERINTIEDLEKANQGRLPLGAFPKQMLGLLQNKQPDLSEKAKIKIEKLIAETKLLSHRLNTKLKSSPKLGKRREKLIKTGVLADWLVKDFMRFQPVVYDAQNQPIKSSKANSTESRLIRRALALYGGEKNRLEGYFKQTNLIGNTNPHPFLNKFNWKACRNLVDFYQQYLEQREKFLEAIKHQPWEPYQYCLLLKVPKENRKNLVKGWEQGGISLPRGLFTEAIRETLSKDLTLSKPIRKEIKKHGRVGFISRAITLYFKEKYQDKHQSFYNLSYKLEAKAPLLKKEEHYEYWQQNKPQSPTESQRLELHTSDRWKDYLLYKRWQHLEKKLRLYRNQDIMLWLMTLELTKNHFKELNLNYHQLKLENLAVNVQEADAKLNPLNQTLPMVLPVKVYPTTAFGEVQYHETPIRTVYIREEQTKALKMGNFKALVKDRHLNGLFSFIKEENDTQKHPISQLRLRRELEIYQSLRVDAFKETLSLEEKLLNKHASLSSLENEFRTLLEEWKKKYAASSMVTDKHIAFIASVRNAFCHNQYPFYKETLHAPILLFTVAQPTTEEKDGLGIAEALLRVLREYCEIVKSQI (SEQ ID NO: 116) Flavobacterium WP_MSSKNESYNKQKTFNHYKQEDKYFFGGFLNNADDNLRQVGKE columnare 063744070FKTRINFNHNNNELASVFKDYFNKEKSVAKREHALNLLSNYFPVLERIQKHTNHNFEQTREIFELLLDTIKKLRDYYTHHYHKPITINPKIYDFLDDTLLDVLITIKKKKVKNDTSRELLKEKLRPELTQLKNQKREELIKKGKKLLEENLENAVFNHCLRPFLEENKTDDKQNKTVSLRKYRKSKPNEETSITLTQSGLVFLMSFFLHRKEFQVFTSGLEGFKAKVNTIKEEKISLNKNNIVYMITHWSYSYYNFKGLKHRIKTDQGVSTLEQNNTTHSLTNTNTKEALLTQIVDYLSKVPNEIYETLSEKQQKEFEEDINEYMRENPENEDSTFSSIVSHKVIRKRYENKFNYFAMRFLDEYAELPTLRFMVNFGDYIKDRQKKILESIQFDSERIIKKEIHLFEKLGLVTEYKKNVYLKETSNIDLSRFPLFPSPSYVMANNNIPFYIDSRSNNLDEYLNQKKKAQSQNRKRNLTFEKYNKEQSKDAIIAMLQKEIGVKDLQQRSTIGLLSCNELPSMLYEVIVKDIKGAELENKIAQKIREQYQSIRDFTLNSPQKDNIPTTLIKTISTDTSVTFENQPIDIPRLKNAIQKELALTQEKLLNVKQHEIEVNNYNRNKNTYKFKNQPKDKVDDNKLQRKYVFYRNEIGQEANWLASDLIHFMKNKSLWKGYMHNELQSFLAFFEDKKNDCIALLETVFNLKEDCILTKDLKNLFLKHGNFIDFYKEYLKLKEDFLNTESTFLENGFIGLPPKILKKELSKRLNYIFIVFQKRQFIIKELEEKKNNLYADAINLSRGIFDEKPTMIPFKKPNPDEFASWFVASYQYNNYQSFYELTPDKIENDKKKKYKNLRAINKVKIQDYYLKLMVDTLYQDLFNQPLDKSLSDFYVSKTDREKIKADAKAYQKRNDSFLWNKVIHLSLQNNRITANPKLKDIGKYKRALQDEKIATLLTYDDRTWTYALQKPEKENENDYKELHYTALNMELQEYEKVRSKKLLKQVQELEKQILDKFYDFSNNATHPEDLEIEDKKGKRHPNFKLYITKALLKNESEIINLENIDIEILIKYYDYNTEKLKEKIKNMDEDEKAKIVNTKENYNKITNVLIKKALVLIIIRNKMAHNQYPPKFIYDLATRFVPKKEEEYFACYFNRVFETITTELWENKKKAKEIV (SEQ ID NO: 117) Riemerella WP_MEKPLPPNVYTLKHKFFWGAFLNIARHNAFITICHINEQLGLTTP anatipestifer 064970887PNDDKIADVVCGTWNNILNNDHDLLKKSQLTELILKHFPFLAAMCYHPPKKEGKKKGSQKEQQKEKENEAQSQAEALNPSELIKVLKTLVKQLRTLRNYYSHHSHKKPDAEKDIFKHLYKAFDASLRMVKEDYKAHFTVNLTQDFAHLNRKGKNKQDNPDFDRYRFEKDGFFTESGLLFFTNLFLDKRDAYWMLKKVSGFKASHKQSEKMTTEVFCRSRILLPKLRLESRYDHNQMLLDMLSELSRYPKLLYEKLSEEDKKRFQVEADGFLDEIEEEQNPFKDTLIRHQDRFPYFALRYLDLNESFKSIRFQVDLGTYHYCIYDKKIGDEQEKRHLTRTLLSFGRLQDFTEINRPQEWKALTKDLDYKETSKQPFISKTTPHYHITDNKIGFRLGTSKELYPSLEVKDGANRIAQYPYNSDFVAHAFISVHELLPLMFYQHLTGKSEDLLKETVRHIQRIYKDFEEERINTIEDLEKANQGRLPLGAFPKQMLGLLQNKQPDLSEKAKIKIEKLIAETKLLSHRLNTKLKSSPKLGKRREKLIKTGVLADWLVKDFMRFQPVAYDAQNQPIESSKANSTEFQLIQRALALYGGEKNRLEGYFKQTNLIGNTNPHPFLNKFNWKACRNLVDFYQQYLEQREKFLEAIKNQPWEPYQYCLLLKIPKENRKNLVKGWEQGGISLPRGLFTEAIRETLSKDLTLSKPIRKEIKKHGRVGFISRAITLYFREKYQDDHQSFYDLPYKLEAKASPLPKKEHYEYWQQNKPQSPTELQRLELHTSDRWKDYLLYKRWQHLEKKLRLYRNQDVMLWLMTLELTKNHFKELNLNYHQLKLENLAVNVQEADAKLNPLNQTLPMVLPVKVYPATAFGEVQYQETPIRTVYIREEQTKALKMGNFKALVKDRRLNGLFSFIKEENDTQKHPISQLRLRRELEIYQSLRVDAFKETLNLEEKLLKKHTSLSSVENKFRILLEEWKKEYAASSMVTDEHIAFIASVRNAFCHNQYPFYEEALHAPIPLFTVAQQTTEEKDGLGIAEALLRVLREYCEIV KSQI (SEQ ID NO: 118

A Table 1A or Table 1B Cas13b effector protein of the invention is, orcomprises, or consists essentially of, or consists of, or involves orrelates to such a protein from or as set forth in above Table 1A orTable 1B or a Cas13b effector protein of an organism set forth in Table1A or Table 1B, as well as those proteins having 90, 91, 92, 93, 94, 9596, 97, 98, 99 or 100 identity therewith, e.g., a protein having one ormore changes within the foregoing percentages wherein the change is,comprises, consists essentially of or consists of a conservativesubstitution groups, wherein conservative substitution may be withreference to the following tabulation:

Class Name of the amino acids Aliphatic Glycine, Alanine, Valine,Leucine, Isoleucine Hydroxyl or Sulfur/ Serine, Cysteine,Selenocysteine, Threonine, Selenium-containing Methionine Cyclic ProlineAromatic Phenylalanine, Tyrosine, Tryptophan Basic Histidine, Lysine,Arginine Acidic and their Amide Aspartate, Glutamate, Asparagine,Glutamine

Or, with reference to knowledge in the art as to a conservativesubstitution, e.g., French et al. “What is a ConservativeSubstitution?”, J. Molecular Evolution, 19(2):171-175 (March 1983);Yampolsky et al., “The Exchangeability of Amino Acids in Proteins,”Genetics 170(4): 1459-1472 (August 2005), doi:10.1534/genetics.104.039107; Gemovic, “Feature-Based Classification ofAmino Acid Substitutions outside Conserved Functional Protein Domains,”The Scientific World Journal, Volume 2013 (2013), Article ID 948617, 10pages dx.doi.org/10.1155/2013/948617. Also, a Table 1A or Table 1BCas13b effector protein of the invention is, or comprises, or consistsessentially of, or consists of, or involves or relates to such a proteinfrom or as set forth in above Table 1A or Table 1B or a Cas13b effectorprotein of an organism set forth in Table 1A or Table 1B, includingmutation, alteration or fusion as herein-discussed, as well as thoseproteins that includes mutation, alteration or fusion asherein-discussed and that either with or without consideration of suchmutation, alteration or fusion has 90, 91, 92, 93, 94, 95 96, 97, 98, 99or 100 identity with that set forth in Table 1A or Table 1B or a Cas13beffector protein of an organism set forth in Table 1A or Table 1B. Inaddition, a Table 1A or Table 1B Cas13b effector protein (that caninclude herein-discussed mutation, alteration or fusion and/or 90, 91,92, 93, 94, 95 96, 97, 98, 99 or 100 identity with that set forth inTable 1A or Table 1B or a Cas13b effector protein of an organism setforth in Table 1A or Table 1B) can be a Cas13b effector protein havingat least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95 96, 97, 98, 99 or100 identity with that set forth in Table 1A or Table 1B or a Cas13beffector protein of an organism set forth in Table 1A or Table 1B and asimilarity, homology or identity to a C2c2 protein of Table 3 asdisplayed between protein(s) of Table 1A or Table 1B and protein(s) ofTable 3. See also FIG. 1 , FIG. 2 .

Example 2: Activity of Cas13b in Eukaryotic Cells

A luciferase targeting assay was performed with different gRNAs directedagainst Gluc. Cas13b orthologues are fused to an NLS or NES oralternatively were not fused to a localization signal. Normalizedprotein expression of luciferase was determined and compared to nontargeting (NT) gRNA. The results for the different Cas13b orthologs areprovided in FIGS. 11 15. In FIG. 16 , the composite data from the mostactive orthologs with the same guide are compiled. It was found that theCas13b orthologs from Bacteroides pyogenes, Prevotella sp. MA2016,Riemerella anatipestifer, Porphyromonas gulae, Porphyromonas gingivalis,and Porphyromonas sp. COT-052OH4946 are particularly active ineukaryotic cells.

Example 3: C2c2 Orthologues

Certain Cas13b orthologs are surprisingly similar to C2c2. FIG. 2provides a tree alignment of C2c2 and Cas13b proteins.

TABLE 2 C2c2 Orthologues Similar to Cas13b Proteins C2c2 orthologue CodeMulti Letter Leptotrichia shahii C2-2 Lsh L wadei F0279 (Lw2) C2-3 Lw2Listeria seeligeri C2-4 Lse Lachnospiraceae bacterium MA2020 C2-5 LbMLachnospiraceae bacterium NK4A179 C2-6 LbNK179 [Clostridium] aminophilumDSM 10710 C2-7 Ca Carnobacterium gallinarum DSM 4847 C2-8 CgCarnobacterium gallinarum DSM 4847 C2-9 Cg2 Paludibacter propionicigenesWB4 C2-10 Pp Listeria weihenstephanensis FSL R9-0317 C2-11 LweiListeriaceae bacterium FSL M6-0635 C2-12 LbFSL Leptotrichia wadei F0279C2-13 Lw Rhodobacter capsulatus SB 1003 C2-14 Rc Rhodobacter capsulatusR121 C2-15 Rc Rhodobacter capsulatus DE442 C2-16 Rc Leptotrichiabuccalis C-1013-b C2-17 Lbu Herbinix hemicellulosilytica C2-18 Hhe[Eubacterium] rectale C2-19 Ere Eubacteriaceae bacterium CHKCI004 C2-20Eba Blautia sp. Marseille-P2398 C2-21 BSm Leptotrichia sp. oral taxon879 str. F0557 C2-22 Lsp Lachnospiraceae bacterium NK4A144 C2-23 NK4A144RNA-binding protein S1 Chloroflexus C2-24 aggregans Demequina aurantiacaC2-25 Thalassospira sp. TSL5-1 C2-26 SAMN04487830_13920Pseudobutyrivibrio C2-27 sp. OR37 SAMN02910398_00008 Butyrivibrio sp.C2-28 YAB3001 Blautia sp. Marseille-P2398 C2-29 Leptotrichia sp.Marseille-P3007 C2-30 Bacteroides ihuae C2-31 SAMN05216357_1045Porphyromonadaceae C2-32 bacterium KH3CP3RA Listeria riparia C2-33Insolitispirillum peregrinum C2-34

The protein sequences of the above C2c2 species are listed in the Table3 below.

TABLE 3 Sequences of C2c2 Species Herein-Identified (See Table 2, Above)c2c2-5 1 Lachnospiraceae MQISKVNHKHVAVGQKDRERITGFIYNDPVGDEKSLEDVVAbacterium KRANDTKVLFNVFNTKDLYDSQESDKSEKDKEIISKGAKFV MA2020AKSFNSAITILKKQNKIYSTLTSQQVIKELKDKFGGARIYDDDIEEALTETLKKSFRKENVRNSIKVLIENAAGIRSSLSKDEEELIQEYFVKQLVEEYTKTKLQKNVVKSIKNQNMVIQPDSDSQVLSLSESRREKQSSAVSSDTLVNCKEKDVLKAFLTDYAVLDEDERNSLLWKLRNLVNLYFYGSESIRDYSYTKEKSVWKEHDEQKANKTLFIDEICHITKIGKNGKEQKVLDYEENRSRCRKQNINYYRSALNYAKNNTSGIFENEDSNHFWIHLIENEVERLYNGIENGEEFKFETGYISEKVWKAVINHLSIKYIALGKAVYNYAMKELSSPGDIEPGKIDDSYINGITSFDYEIIKAEESLQRDISMNVVFATNYLACATVDTDKDFLLFSKEDIRSCTKKDGNLCKNIMQFWGGYSTWKNFCEEYLKDDKDALELLYSLKSMLYSMRNSSFHFSTENVDNGSWDTELIGKLFEEDCNRAARIEKEKFYNNNLHMFYSSSLLEKVLERLYSSHHERASQVPSFNRVFVRKNFPSSLSEQRITPKFTDSKDEQIWQSAVYYLCKEIYYNDFLQSKEAYKLFREGVKNLDKNDINNQKAADSFKQAVVYYGKAIGNATLSQVCQAIMTEYNRQNNDGLKKKSAYAEKQNSNKYKHYPLFLKQVLQSAFWEYLDENKEIYGFISAQIHKSNVEIKAEDFIANYSSQQYKKLVDKVKKTPELQKWYTLGRLINPRQANQFLGSIRNYVQFVKDIQRRAKENGNPIRNYYEVLESDSIIKILEMCTKLNGTTSNDIHDYFRDEDEYAEYISQFVNFGDVHSGAALNAFCNSESEGKKNGIYYDGINPIVNRNWVLCKLYGSPDLISKIISRVNENMIHDFHKQEDLIREYQIKGICSNKKEQQDLRTFQVLKNRVELRDIVEYSEIINELYGQLIKWCYLRERDLMYFQLGFHYLCLNNASSKEADYIKINVDDRNISGAILYQIAAMYINGLPVYYKKDDMYVALKSGKKASDELNSNEQTSKKINYFLKYGNNILGDKKDQLYLAGLELFENVAEHENIIIFRNEIDHFHYFYDRDRSMLDLYSEVFDRFFTYDMKLRKNVVNMLYNILLDHNIVSSFVFETGEKKVGRGDSEVIKPSAKIRLRANNGVSSDVFTYKVGSKDELKIATLPAKNEEFLLNVARLIYYPDMEAVSENMVREGVVKVEKSNDKKGKISRGSNTRSSNQSKYNNKSKNRMNYSMGSIFEKMDLK FD (SEQ ID NO: 119) c2c2-6 2Lachnospiraceae MKISKVREENRGAKLTVNAKTAVVSENRSQEGILYNDPSRY bacteriumGKSRKNDEDRDRYIESRLKSSGKLYRIFNEDKNKRETDELQ NK4A179WFLSEIVKKINRRNGLVLSDMLSVDDRAFEKAFEKYAELSYTNRRNKVSGSPAFETCGVDAATAERLKGIISETNFINRIKNNIDNKVSEDIIDRIIAKYLKKSLCRERVKRGLKKLLMNAFDLPYSDPDIDVQRDFIDYVLEDFYHVRAKSQVSRSIKNMNMPVQPEGDGKFAITVSKGGTESGNKRSAEKEAFKKFLSDYASLDERVRDDMLRRMRRLVVLYFYGSDDSKLSDVNEKFDVWEDHAARRVDNREFIKLPLENKLANGKTDKDAERIRKNTVKELYRNQNIGCYRQAVKAVEEDNNGRYFDDKMLNMFFIHRIEYGVEKIYANLKQVTEFKARTGYLSEKIWKDLINYISIKYIAMGKAVYNYAMDELNASDKKEIELGKISEEYLSGISSFDYELIKAEEMLQRETAVYVAFAARHLSSQTVELDSENSDFLLLKPKGTMDKNDKNKLASNNILNFLKDKETLRDTILQYFGGHSLWTDFPFDKYLAGGKDDVDFLTDLKDVIYSMRNDSFHYATENHNNGKWNKELISAMFEHETERMTVVMKDKFYSNNLPMFYKNDDLKKLLIDLYKDNVERASQVPSFNKVFVRKNFPALVRDKDNLGIELDLKADADKGENELKFYNALYYMFKEIYYNAFLNDKNVRERFITKATKVADNYDRNKERNLKDRIKSAGSDEKKKLREQLQNYIAENDFGQRIKNIVQVNPDYTLAQICQLIMTEYNQQNNGCMQKKSAARKDINKDSYQHYKMLLLVNLRKAFLEFIKENYAFVLKPYKHDLCDKADFVPDFAKYVKPYAGLISRVAGSSELQKWYIVSRFLSPAQANHMLGFLHSYKQYVWDIYRRASETGTEINHSIAEDKIAGVDITDVDAVIDLSVKLCGTISSEISDYFKDDEVYAEYISSYLDFEYDGGNYKDSLNRFCNSDAVNDQKVALYYDGEHPKLNRNIILSKLYGERRFLEKITDRVSRSDIVEYYKLKKETSQYQTKGIFDSEDEQKNIKKFQEMKNIVEFRDLMDYSEIADELQGQLINWIYLRERDLMNFQLGYHYACLNNDSNKQATYVTLDYQGKKNRKINGAILYQICAMYINGLPLYYVDKDSSEWTVSDGKESTGAKIGEFYRYAKSFENTSDCYASGLEIFENISEHDNITELRNYIEHFRYYSSFDRSFLGIYSEVFDRFFTYDLKYRKNVPTILYNILLQHFVNVRFEFVSGKKMIGIDKKDRKIAKEKECARITIREKNGVYSEQFTYKLKNGTVYVDARDKRYLQSIIRLLFYPEKVNMDEMIEVKEKKKPSDNNTGKGYSKRDRQQDRKEYDKYKEKKKKEGNFLSGMGGNINWDEINAQLKN (SEQ ID NO: 120) c2c2-7 3 [Clostridium]MKFSKVDHTRSAVGIQKATDSVHGMLYTDPKKQEVNDLDK aminophilumRFDQLNVKAKRLYNVFNQSKAEEDDDEKRFGKVVKKLNRE DSMLKDLLFHREVSRYNSIGNAKYNYYGIKSNPEEIVSNLGMVES 10710LKGERDPQKVISKLLLYYLRKGLKPGTDGLRMILEASCGLRKLSGDEKELKVFLQTLDEDFEKKTFKKNLIRSIENQNMAVQPSNEGDPIIGITQGRFNSQKNEEKSAIERMMSMYADLNEDHREDVLRKLRRLNVLYFNVDTEKTEEPTLPGEVDTNPVFEVWHDHEKGKENDRQFATFAKILTEDRETRKKEKLAVKEALNDLKSAIRDHNIMAYRCSIKVTEQDKDGLFFEDQRINRFWIHHIESAVERILASINPEKLYKLRIGYLGEKVWKDLLNYLSIKYIAVGKAVFHFAMEDLGKTGQDIELGKLSNSVSGGLTSFDYEQIRADETLQRQLSVEVAFAANNLFRAVVGQTGKKIEQSKSEENEEDFLLWKAEKIAESIKKEGEGNTLKSILQFFGGASSWDLNHFCAAYGNESSALGYETKFADDLRKAIYSLRNETFHFTTLNKGSFDWNAKLIGDMFSHEAATGIAVERTRFYSNNLPMFYRESDLKRIMDHLYNTYHPRASQVPSFNSVFVRKNFRLFLSNTLNTNTSFDTEVYQKWESGVYYLFKEIYYNSFLPSGDAHHLFFEGLRRIRKEADNLPIVGKEAKKRNAVQDFGRRCDELKNLSLSAICQMIMTEYNEQNNGNRKVKSTREDKRKPDIFQHYKMLLLRTLQEAFAIYIRREEFKFIFDLPKTLYVMKPVEEFLPNWKSGMFDSLVERVKQSPDLQRWYVLCKFLNGRLLNQLSGVIRSYIQFAGDIQRRAKANHNRLYMDNTQRVEYYSNVLEVVDFCIKGTSRFSNVFSDYFRDEDAYADYLDNYLQFKDEKIAEVSSFAALKTFCNEEEVKAGIYMDGENPVMQRNIVMAKLFGPDEVLKNVVPKVTREEIEEYYQLEKQIAPYRQNGYCKSEEDQKKLLRFQRIKNRVEFQTITEFSEIINELLGQLISWSFLRERDLLYFQLGFHYLCLHNDTEKPAEYKEISREDGTVIRNAILHQVAAMYVGGLPVYTLADKKLAAFEKGEADCKLSISKDTAGAGKKIKDFFRYSKYVLIKDRMLTDQNQKYTIYLAGLELFENTDEHDNITDVRKYVDHFKYYATSDENAMSILDLYSEIHDRFFTYDMKYQKNVANMLENILLRHFVLIRPEFFTGSKKVGEGKKITCKARAQIEIAENGMRSEDFTYKLSDGKKNISTCMIAARDQKYLNTVARLLYYPHEAKKSIVDTREKKNNKKTNRGDGTFNKQKGTARKEKDNGPREFNDTGF SNTPFAGFDPFRNS (SEQ ID NO: 121)c2c2-8 5 Carnobacterium MRITKVKIKLDNKLYQVTMQKEEKYGTLKLNEESRKSTAEILgallinarum RLKKASFNKSFHSKTINSQKENKNATIKKNGDYISQIFEKLVG DSM 4847VDTNKNIRKPKMSLTDLKDLPKKDLALFIKRKFKNDDIVEIKNLDLISLFYNALQKVPGEHFTDESWADFCQEMMPYREYKNKFIERKIILLANSIEQNKGFSINPETFSKRKRVLHQWAIEVQERGDFSILDEKLSKLAEIYNFKKMCKRVQDELNDLEKSMKKGKNPEKEKEAYKKQKNFKIKTIWKDYPYKTHIGLIEKIKENEELNQFNIEIGKYFEHYFPIKKERCTEDEPYYLNSETIATTVNYQLKNALISYLMQIGKYKQFGLENQVLDSKKLQEIGIYEGFQTKFMDACVFATSSLKNIIEPMRSGDILGKREFKEAIATSSFVNYHHFFPYFPFELKGMKDRESELIPFGEQTEAKQMQNIWALRGSVQQIRNEIFHSFDKNQKFNLPQLDKSNFEFDASENSTGKSQSYIETDYKFLFEAEKNQLEQFFIERIKSSGALEYYPLKSLEKLFAKKEMKFSLGSQVVAFAPSYKKLVKKGHSYQTATEGTANYLGLSYYNRYELKEESFQAQYYLLKLIYQYVFLPNFSQGNSPAFRETVKAILRINKDEARKKMKKNKKFLRKYAFEQVREMEFKETPDQYMSYLQSEMREEKVRKAEKNDKGFEKNITMNFEKLLMQIFVKGFDVFLTTFAGKELLLSSEEKVIKETEISLSKKINEREKTLKASIQVEHQLVATNSAISYWLFCKLLDSRHLNELRNEMIKFKQSRIKFNHTQHAELIQNLLPIVELTILSNDYDEKNDSQNVDVSAYFEDKSLYETAPYVQTDDRTRVSFRPILKLEKYHTKSLIEALLKDNPQFRVAATDIQEWMHKREEIGELVEKRKNLHTEWAEGQQTLGAEKREEYRDYCKKIDRFNWKANKVTLTYLSQLHYLITDLLGRMVGFSALFERDLVYFSRSFSELGGETYHISDYKNLSGVLRLNAEVKPIKIKNIKVIDNEENPYKGNEPEVKPFLDRLHAYLENVIGIKAVHGKIRNQTAHLSVLQLELSMIESMNNLRDLMAYDRKLKNAVTKSMIKILDKHGMILKLKIDENHKNFEIESLIPKEIIHLKDKAIKTNQVSEEYCQLVLALLTTNPGNQLN (SEQ ID NO: 122) c2c2-9 6Carnobacterium MRMTKVKINGSPVSMNRSKLNGHLVWNGTTNTVNILTKKE gallinarumQSFAASFLNKTLVKADQVKGYKVLAENIFIIFEQLEKSNSEKP DSM 4847SVYLNNIRRLKEAGLKRFFKSKYHEEIKYTSEKNQSVPTKLNLIPLFFNAVDRIQEDKFDEKNWSYFCKEMSPYLDYKKSYLNRKKEILANSIQQNRGFSMPTAEEPNLLSKRKQLFQQWAMKFQESPLIQQNNFAVEQFNKEFANKINELAAVYNVDELCTAITEKLMNFDKDKSNKTRNFEIKKLWKQHPHNKDKALIKLFNQEGNEALNQFNIELGKYFEHYFPKTGKKESAESYYLNPQTIIKTVGYQLRNAFVQYLLQVGKLHQYNKGVLDSQTLQEIGMYEGFQTKFMDACVFASSSLRNIIQATTNEDILTREKFKKELEKNVELKHDLFFKTEIVEERDENPAKKIAMTPNELDLWAIRGAVQRVRNQIFHQQINKRHEPNQLKVGSFENGDLGNVSYQKTIYQKLFDAEIKDIEIYFAEKIKSSGALEQYSMKDLEKLFSNKELTLSLGGQVVAFAPSYKKLYKQGYFYQNEKTIELEQFTDYDFSNDVFKANYYLIKLIYHYVFLPQFSQANNKLFKDTVHYVIQQNKELNTTEKDKKNNKKIRKYAFEQVKLMKNESPEKYMQYLQREMQEERTIKEAKKTNEEKPNYNFEKLLIQIFIKGFDTFLRNFDLNLNPAEELVGTVKEKAEGLRKRKERIAKILNVDEQIKTGDEEIAFWIFAKLLDARHLSELRNEMIKFKQSSVKKGLIKNGDLIEQMQPILELCILSNDSESMEKESFDKIEVFLEKVELAKNEPYMQEDKLTPVKFRFMKQLEKYQTRNFIENLVIENPEFKVSEKIVLNWHEEKEKIADLVDKRTKLHEEWASKAREIEEYNEKIKKNKSKKLDKPAEFAKFAEYKIICEAIENFNRLDHKVRLTYLKNLHYLMIDLMGRMVGFSVLFERDFVYMGRSYSALKKQSIYLNDYDTFANIRDWEVNENKHLFGTSSSDLTFQETAEFKNLKKPMENQLKALLGVTNHSFEIRNNIAHLHVLRNDGKGEGVSLLSCMNDLRKLMSYDRKLKNAVTKAIIKILDKHGMILKLTNNDHTKPFEIESLKPKKIIHLEKSNHSFPMDQVSQEYCDLVKKMLVFTN (SEQ ID NO: 123) c2c2- 7Paludibacter MRVSKVKVKDGGKDKMVLVHRKTTGAQLVYSGQPVSNET 10 propionicigenesSNILPEKKRQSFDLSTLNKTIIKFDTAKKQKLNVDQYKIVEKI WB4FKYPKQELPKQIKAEEILPFLNHKFQEPVKYWKNGKEESFNLTLLIVEAVQAQDKRKLQPYYDWKTWYIQTKSDLLKKSIENNRIDLTENLSKRKKALLAWETEFTASGSIDLTHYHKVYMTDVLCKMLQDVKPLTDDKGKINTNAYHRGLKKALQNHQPAIFGTREVPNEANRADNQLSIYHLEVVKYLEHYFPIKTSKRRNTADDIAHYLKAQTLKTTIEKQLVNAIRANIIQQGKTNHHELKADTTSNDLIRIKTNEAFVLNLTGTCAFAANNIRNMVDNEQTNDILGKGDFIKSLLKDNTNSQLYSFFFGEGLSTNKAEKETQLWGIRGAVQQIRNNVNHYKKDALKTVFNISNFENPTITDPKQQTNYADTIYKARFINELEKIPEAFAQQLKTGGAVSYYTIENLKSLLTTFQFSLCRSTIPFAPGFKKVFNGGINYQNAKQDESFYELMLEQYLRKENFAEESYNARYFMLKLIYNNLFLPGFTTDRKAFADSVGFVQMQNKKQAEKVNPRKKEAYAFEAVRPMTAADSIADYMAYVQSELMQEQNKKEEKVAEETRINFEKFVLQVFIKGFDSFLRAKEFDFVQMPQPQLTATASNQQKADKLNQLEASITADCKLTPQYAKADDATHIAFYVFCKLLDAAHLSNLRNELIKFRESVNEFKFHHLLEIIEICLLSADVVPTDYRDLYSSEADCLARLRPFIEQGADITNWSDLFVQSDKHSPVIHANIELSVKYGTTKLLEQIINKDTQFKTTEANFTAWNTAQKSIEQLIKQREDHHEQWVKAKNADDKEKQERKREKSNFAQKFIEKHGDDYLDICDYINTYNWLDNKMHFVHLNRLHGLTIELLGRMAGFVALFDRDFQFFDEQQIADEFKLHGFVNLHSIDKKLNEVPTKKIKEIYDIRNKIIQINGNKINESVRANLIQFISSKRNYYNNAFLHVSNDEIKEKQMYDIRNHIAHFNYLTKDAADFSLIDLINELRELLHYDRKLKNAVSKAFIDLFDKHGMILKLKLNADHKLKVESLEPKKIYHLGSSAKDKPEYQYCTNQVMMAYCNMCRSLLEMKK (SEQ ID NO: 124) c2c2- 9 ListeriaMLALLHQEVPSQKLHNLKSLNTESLTKLFKPKFQNMISYPPS 11 weihenstephanensisKGAEHVQFCLTDIAVPAIRDLDEIKPDWGIFFEKLKPYTDWA FSL R9-ESYIHYKQTTIQKSIEQNKIQSPDSPRKLVLQKYVTAFLNGEP 0317LGLDLVAKKYKLADLAESFKVVDLNEDKSANYKIKACLQQHQRNILDELKEDPELNQYGIEVKKYIQRYFPIKRAPNRSKHARADFLKKELIESTVEQQFKNAVYHYVLEQGKMEAYELTDPKTKDLQDIRSGEAFSFKFINACAFASNNLKMILNPECEKDILGKGDFKKNLPNSTTQSDVVKKMIPFFSDEIQNVNFDEAIWAIRGSIQQIRNEVYHCKKHSWKSILKIKGFEFEPNNMKYTDSDMQKLMDKDIAKIPDFIEEKLKSSGIIRFYSHDKLQSIWEMKQGFSLLTTNAPFVPSFKRVYAKGHDYQTSKNRYYDLGLTTFDILEYGEEDFRARYFLTKLVYYQQFMPWFTADNNAFRDAANFVLRLNKNRQQDAKAFINIREVEEGEMPRDYMGYVQGQIAIHEDSTEDTPNHFEKFISQVFIKGFDSHMRSADLKFIKNPRNQGLEQSEIEEMSFDIKVEPSFLKNKDDYIAFWTFCKMLDARHLSELRNEMIKYDGHLTGEQEIIGLALLGVDSRENDWKQFFSSEREYEKIMKGYVGEELYQREPYRQSDGKTPILFRGVEQARKYGTETVIQRLFDASPEFKVSKCNITEWERQKETIEETIERRKELHNEWEKNPKKPQNNAFFKEYKECCDAIDAYNWHKNKTTLVYVNELHHLLIEILGRYVGYVAIADRDFQCMANQYFKHSGITERVEYWGDNRLKSIKKLDTFLKKEGLFVSEKNARNHIAHLNYLSLKSECTLLYLSERLREIFKYDRKLKNAVSKSLIDILDRHGMSVVFANLKENKHRLVIKSLEPKKLRHLGEKKIDNGYIETNQVSEEY CGIVKRLLEI (SEQ ID NO: 125)c2c2- 10 Listeriaceae MKITKMRVDGRTIVMERTSKEGQLGYEGIDGNKTTEIIFDKK 12bacterium KESFYKSILNKTVRKPDEKEKNRRKQAINKAINKEITELMLA FSL M6-VLHQEVPSQKLHNLKSLNTESLTKLFKPKFQNMISYPPSKGA 0635 =EHVQFCLTDIAVPAIRDLDEIKPDWGIFFEKLKPYTDWAESYI ListeriaHYKQTTIQKSIEQNKIQSPDSPRKLVLQKYVTAFLNGEPLGL newyorkensisDLVAKKYKLADLAESFKLVDLNEDKSANYKIKACLQQHQR FSLNILDELKEDPELNQYGIEVKKYIQRYFPIKRAPNRSKHARADF M6-0635LKKELIESTVEQQFKNAVYHYVLEQGKMEAYELTDPKTKDLQDIRSGEAFSFKFINACAFASNNLKMILNPECEKDILGKGNFKKNLPNSTTRSDVVKKMIPFFSDELQNVNFDEAIWAIRGSIQQIRNEVYHCKKHSWKSILKIKGFEFEPNNMKYADSDMQKLMDKDIAKIPEFIEEKLKSSGVVRFYRHDELQSIWEMKQGFSLLTTNAPFVPSFKRVYAKGHDYQTSKNRYYNLDLTTFDILEYGEEDFRARYFLTKLVYYQQFMPWFTADNNAFRDAANFVLRLNKNRQQDAKAFINIREVEEGEMPRDYMGYVQGQIAIHEDSIEDTPNHFEKFISQVFIKGFDRHMRSANLKFIKNPRNQGLEQSEIEEMSFDIKVEPSFLKNKDDYIAFWIFCKMLDARHLSELRNEMIKYDGHLTGEQEIIGLALLGVDSRENDWKQFFSSEREYEKIMKGYVVEELYQREPYRQSDGKTPILFRGVEQARKYGTETVIQRLFDANPEFKVSKCNLAEWERQKETIEETIKRRKELHNEWAKNPKKPQNNAFFKEYKECCDAIDAYNWHKNKTTLAYVNELHHLLIEILGRYVGYVAIADRDFQCMANQYFKHSGITERVEYWGDNRLKSIKKLDTFLKKEGLFVSEKNARNHIAHLNYLSLKSECTLLYLSERLREIFKYDRKLKNAVSKSLIDILDRHGMSVVFANLKENKHRLVIKSLEPKKLRHLGGKKIDGGYIETNQVSEEYCGI VKRLLEM (SEQ ID NO: 126)c2c2- 12 Leptotrichia MKVTKVDGISHKKYIEEGKLVKSTSEENRTSERLSELLSIRLD 13wadei IYIKNPDNASEEENRIRRENLKKFFSNKVLHLKDSVLYLKNR F0279KEKNAVQDKNYSEEDISEYDLKNKNSFSVLKKILLNEDVNSEELEIFRKDVEAKLNKINSLKYSFEENKANYQKINENNVEKVGGKSKRNIIYDYYRESAKRNDYINNVQEAFDKLYKKEDIEKLFFLIENSKKHEKYKIREYYHKIIGRKNDKENFAKIIYEEIQNVNNIKELIEKIPDMSELKKSQVFYKYYLDKEELNDKNIKYAFCHFVEIEMSQLLKNYVYKRLSNISNDKIKRIFEYQNLKKLIENKLLNKLDTYVRNCGKYNYYLQVGEIATSDFIARNRQNEAFLRNIIGVSSVAYFSLRNILETENENDITGRMRGKTVKNNKGEEKYVSGEVDKIYNENKQNEVKENLKMFYSYDFNMDNKNEIEDFFANIDEAISSIRHGIVHFNLELEGKDIFAFKNIAPSEISKKMFQNEINEKKLKLKIFKQLNSANVFNYYEKDVIIKYLKNTKFNFVNKNIPFVPSFTKLYNKIEDLRNTLKFFWSVPKDKEEKDAQIYLLKNIYYGEFLNKFVKNSKVFFKITNEVIKINKQRNQKTGHYKYQKFENIEKTVPVEYLAIIQSREMINNQDKEEKNTYIDFIQQIFLKGFIDYLNKNNLKYIESNNNNDNNDIFSKIKIKKDNKEKYDKILKNYEKHNRNKEIPHEINEFVREIKLGKILKYTENLNMFYLILKLLNHKELTNLKGSLEKYQSANKEETFSDELELINLLNLDNNRVTEDFELEANEIGKFLDFNENKIKDRKELKKFDTNKIYFDGENIIKHRAFYNIKKYGMLNLLEKIADKAKYKISLKELKEYSNKKNEIEKNYTMQQNLHRKYARPKKDEKFNDEDYKEYEKAIGNIQKYTHLKNKVEFNELNLLQGLLLKILHRLVGYTSIWERDLRFRLKGEFPENHYIEEIFNFDNSKNVKYKSGQIVEKYINFYKELYKDNVEKRSIYSDKKVKKLKQEKKDLYIRNYIAHFNYIPHAEISLLEVLENLRKLLSYDRKLKNAIMKSIVDILKEYGFVATFKIGADKKIEIQTLESEKIVHLKNLKKKKLMTDRNSEELCELVK VMFEYKALE (SEQ ID NO: 127)c2c2- 15 Rhodobacter MQIGKVQGRTISEFGDPAGGLKRKISTDGKNRKELPAHLSSD 14capsulatus PKALIGQWISGIDKIYRKPDSRKSDGKAIHSPTPSKMQFDARD SB 1003DLGEAFWKLVSEAGLAQDSDYDQFKRRLHPYGDKFQPADSGAKLKFEADPPEPQAFHGRWYGAMSKRGNDAKELAAALYEHLHVDEKRIDGQPKRNPKTDKFAPGLVVARALGIESSVLPRGMARLARNWGEEEIQTYFVVDVAASVKEVAKAAVSAAQAFDPPRQVSGRSLSPKVGFALAEHLERVTGSKRCSFDPAAGPSVLALHDEVKKTYKRLCARGKNAARAFPADKTELLALMRHTHENRVRNQMVRMGRVSEYRGQQAGDLAQSHYWTSAGQTEIKESEIFVRLWVGAFALAGRSMKAWIDPMGKIVNTEKNDRDLTAAVNIRQVISNKEMVAEAMARRGIYFGETPELDRLGAEGNEGFVFALLRYLRGCRNQTFHLGARAGFLKEIRKELEKTRWGKAKEAEHVVLTDKTVAAIRAIIDNDAKALGARLLADLSGAFVAHYASKEHFSTLYSEIVKAVKDAPEVSSGLPRLKLLLKRADGVRGYVHGLRDTRKHAFATKLPPPPAPRELDDPATKARYIALLRLYDGPFRAYASGITGTALAGPAARAKEAATALAQSVNVTKAYSDVMEGRTSRLRPPNDGETLREYLSALTGETATEFRVQIGYESDSENARKQAEFIENYRRDMLAFMFEDYIRAKGFDWILKIEPGATAMTRAPVLPEPIDTRGQYEHWQAALYLVMHFVPASDVSNLLHQLRKWEALQGKYELVQDGDATDQADARREALDLVKRFRDVLVLFLKTGEARFEGRAAPFDLKPFRALFANPATFDRLFMATPTTARPAEDDPEGDGASEPELRVARTLRGLRQIARYNHMAVLSDLFAKHKVRDEEVARLAEIEDETQEKSQIVAAQELRTDLHDKVMKCHPKTISPEERQSYAAAIKTIEEHRFLVGRVYLGDHLRLHRLMMDVIGRLIDYAGAYERDTGTFLINASKQLGAGADWAVTIAGAANTDARTQTRKDLAHFNVLDRADGTPDLTALVNRAREMMAYDRKRKNAVPRSILDMLARLGLTLKWQMKDHLLQDATITQAAIKHLDKVRLTVGGPAAVTEARFSQDYLQMVAAVFNGSVQNPKPRRRDDGDAWHKPPKPATAQSQPDQKPPNKAPSAGSRLPPPQVGEVYEGVVVKVIDTGSLGFLAVEGVAGNIGLHISRLRRIREDAIIVGRRYRFRVEIYVPPKSNTSKL NAADLVRID (SEQ ID NO: 128)c2c2- 16 Rhodobacter MQIGKVQGRTISEFGDPAGGLKRKISTDGKNRKELPAHLSSD 15capsulatus PKALIGQWISGIDKIYRKPDSRKSDGKAIHSPTPSKMQFDARD R121DLGEAFWKLVSEAGLAQDSDYDQFKRRLHPYGDKFQPADSGAKLKFEADPPEPQAFHGRWYGAMSKRGNDAKELAAALYEHLHVDEKRIDGQPKRNPKTDKFAPGLVVARALGIESSVLPRGMARLARNWGEEEIQTYFVVDVAASVKEVAKAAVSAAQAFDPPRQVSGRSLSPKVGFALAEHLERVTGSKRCSFDPAAGPSVLALHDEVKKTYKRLCARGKNAARAFPADKTELLALMRHTHENRVRNQMVRMGRVSEYRGQQAGDLAQSHYWTSAGQTEIKESEIFVRLWVGAFALAGRSMKAWIDPMGKIVNTEKNDRDLTAAVNIRQVISNKEMVAEAMARRGIYFGETPELDRLGAEGNEGFVFALLRYLRGCRNQTFHLGARAGFLKEIRKELEKTRWGKAKEAEHVVLTDKTVAAIRAIIDNDAKALGARLLADLSGAFVAHYASKEHFSTLYSEIVKAVKDAPEVSSGLPRLKLLLKRADGVRGYVHGLRDTRKHAFATKLPPPPAPRELDDPATKARYIALLRLYDGPFRAYASGITGTALAGPAARAKEAATALAQSVNVTKAYSDVMEGRSSRLRPPNDGETLREYLSALTGETATEFRVQIGYESDSENARKQAEFIENYRRDMLAFMFEDYIRAKGFDWILKIEPGATAMTRAPVLPEPIDTRGQYEHWQAALYLVMHFVPASDVSNLLHQLRKWEALQGKYELVQDGDATDQADARREALDLVKRFRDVLVLFLKTGEARFEGRAAPFDLKPFRALFANPATFDRLFMATPTTARPAEDDPEGDGASEPELRVARTLRGLRQIARYNHMAVLSDLFAKHKVRDEEVARLAEIEDETQEKSQIVAAQELRTDLHDKVMKCHPKTISPEERQSYAAAIKTIEEHRFLVGRVYLGDHLRLHRLMMDVIGRLIDYAGAYERDTGTFLINASKQLGAGADWAVTIAGAANTDARTQTRKDLAHFNVLDRADGTPDLTALVNRAREMMAYDRKRKNAVPRSILDMLARLGLTLKWQMKDHLLQDATITQAAIKHLDKVRLTVGGPAAVTEARFSQDYLQMVAAVFNGSVQNPKPRRRDDGDAWHKPPKPATAQSQPDQKPPNKAPSAGSRLPPPQVGEVYEGVVVKVIDTGSLGFLAVEGVAGNIGLHISRLRRIREDAIIVGRRYRFRVEIYVPPKSNTSKL NAADLVRID (SEQ ID NO: 129)c2c2- 17 Rhodobacter MQIGKVQGRTISEFGDPAGGLKRKISTDGKNRKELPAHLSSD 16capsulatus PKALIGQWISGIDKIYRKPDSRKSDGKAIHSPTPSKMQFDARD DE442DLGEAFWKLVSEAGLAQDSDYDQFKRRLHPYGDKFQPADSGAKLKFEADPPEPQAFHGRWYGAMSKRGNDAKELAAALYEHLHVDEKRIDGQPKRNPKTDKFAPGLVVARALGIESSVLPRGMARLARNWGEEEIQTYFVVDVAASVKEVAKAAVSAAQAFDPPRQVSGRSLSPKVGFALAEHLERVTGSKRCSFDPAAGPSVLALHDEVKKTYKRLCARGKNAARAFPADKTELLALMRHTHENRVRNQMVRMGRVSEYRGQQAGDLAQSHYWTSAGQTEIKESEIFVRLWVGAFALAGRSMKAWIDPMGKIVNTEKNDRDLTAAVNIRQVISNKEMVAEAMARRGIYFGETPELDRLGAEGNEGFVFALLRYLRGCRNQTFHLGARAGFLKEIRKELEKTRWGKAKEAEHVVLTDKTVAAIRAIIDNDAKALGARLLADLSGAFVAHYASKEHFSTLYSEIVKAVKDAPEVSSGLPRLKLLLKRADGVRGYVHGLRDTRKHAFATKLPPPPAPRELDDPATKARYIALLRLYDGPFRAYASGITGTALAGPAARAKEAATALAQSVNVTKAYSDVMEGRSSRLRPPNDGETLREYLSALTGETATEFRVQIGYESDSENARKQAEFIENYRRDMLAFMFEDYIRAKGFDWILKIEPGATAMTRAPVLPEPIDTRGQYEHWQAALYLVMHFVPASDVSNLLHQLRKWEALQGKYELVQDGDATDQADARREALDLVKRFRDVLVLFLKTGEARFEGRAAPFDLKPFRALFANPATFDRLFMATPTTARPAEDDPEGDGASEPELRVARTLRGLRQIARYNHMAVLSDLFAKHKVRDEEVARLAEIEDETQEKSQIVAAQELRTDLHDKVMKCHPKTISPEERQSYAAAIKTIEEHRFLVGRVYLGDHLRLHRLMMDVIGRLIDYAGAYERDTGTFLINASKQLGAGADWAVTIAGAANTDARTQTRKDLAHFNVLDRADGTPDLTALVNRAREMMAYDRKRKNAVPRSILDMLARLGLTLKWQMKDHLLQDATITQAAIKHLDKVRLTVGGPAAVTEARFSQDYLQMVAAVFNGSVQNPKPRRRDDGDAWHKPPKPATAQSQPDQKPPNKAPSAGSRLPPPQVGEVYEGVVVKVIDTGSLGFLAVEGVAGNIGLHISRLRRIREDAIIVGRRYRFRVEIYVPPKSNTSKL NAADLVRID (SEQ ID NO: 130)c2-2 MGNLFGHKRWYEVRDKKDFKIKRKVKVKRNYDGNKYILNINENNNKEKIDNNKFIRKYINYKKNDNILKEFTRKFHAGNILFKLKGKEGIIRIENNDDFLETEEVVLYIEAYGKSEKLKALGITKKKIIDEAIRQGITKDDKKIEIKRQENEEEIEIDIRDEYTNKTLNDCSIILRIIENDELETKKSIYEIFKNINMSLYKIIEKIIENETEKVFENRYYEEHLREKLLKDDKIDVILTNFMEIREKIKSNLEILGFVKFYLNVGGDKKKSKNKKMLVEKILNINVDLTVEDIADFVIKELEFWNITKRIEKVKKVNNEFLEKRRNRTYIKSYVLLDKHEKFKIERENKKDKIVKFFVENIKNNSIKEKIEKILAEFKIDELIKKLEKELKKGNCDTEIFGIFKKHYKVNFDSKKFSKKSDEEKELYKIIYRYLKGRIEKILVNEQKVRLKKMEKIEIEKILNESILSEKILKRVKQYTLEHIMYLGKLRHNDIDMTTVNTDDFSRLHAKEELDLELITFFASTNMELNKIFSRENINNDENIDFFGGDREKNYVLDKKILNSKIKIIRDLDFIDNKNNITNNFIRKFTKIGTNERNRILHAISKERDLQGTQDDYNKVINIIQNLKISDEEVSKALNLDVVFKDKKNIITKINDIKISEENNNDIKYLPSFSKVLPEILNLYRNNPKNEPFDTIETEKIVLNALIYVNKELYKKLILEDDLEENESKNIFLQELKKTLGNIDEIDENIIENYYKNAQISASKGNNKAIKKYQKKVIECYIGYLRKNYEELFDFSDFKMNIQEIKKQIKDINDNKTYERITVKTSDKTIVINDDFEYIISIFALLNSNAVINKIRNRFFATSVWLNTSEYQNIIDILDEIMQLNTLRNECITENWNLNLEEFIQKMKEIEKDFDDFKIQTKKEIFNNYYEDIKNNILTEFKDDINGCDVLEKKLEKIVIFDDETKFEIDKKSNILQDEQRKLSNINKKDLKKKVDQYIKDKDQEIKSKILCRIIFNSDFLKKYKKEIDNLIEDMESENENKFQEIYYPKERKNELYIYKKNLFLNIGNPNFDKIYGLISNDIKMADAKFLFNIDGKNIRKNKISEIDAILKNLNDKLNGYSKEYKEKYIKKLKENDDFFAKNIQNKNYKSFEKDYNRVSEYKKIRDLVEFNYLNKIESYLIDINWKLAIQMARFERDMHYIVNGLRELGIIKLSGYNTGISRAYPKRNGSDGFYTTTAYYKFFDEESYKKFEKICYGFGIDLSENSEINKPENESIRNYISHFYIVRNPFADYSIAEQIDRVSNLLSYSTRYNNSTYASVFEVFKKDVNLDYDELKKKFKLIGNNDILERLMKPKKVSVLELESYNSDYIKNLIIELLTKI ENTNDTL (SEQ ID NO: 131)c2-3 L wadei MKVTKVDGISHKKYIEEGKLVKSTSEENRTSERLSELLSIRLD (Lw2)IYIKNPDNASEEENRIRRENLKKFFSNKVLHLKDSVLYLKNRKEKNAVQDKNYSEEDISEYDLKNKNSFSVLKKILLNEDVNSEELEIFRKDVEAKLNKINSLKYSFEENKANYQKINENNVEKVGGKSKRNIIYDYYRESAKRNDYINNVQEAFDKLYKKEDIEKLFFLIENSKKHEKYKIREYYHKIIGRKNDKENFAKIIYEEIQNVNNIKELIEKIPDMSELKKSQVFYKYYLDKEELNDKNIKYAFCHFVEIEMSQLLKNYVYKRLSNISNDKIKRIFEYQNLKKLIENKLLNKLDTYVRNCGKYNYYLQVGEIATSDFIARNRQNEAFLRNIIGVSSVAYFSLRNILETENENDITGRMRGKTVKNNKGEEKYVSGEVDKIYNENKQNEVKENLKMFYSYDFNMDNKNEIEDFFANIDEAISSIRHGIVHFNLELEGKDIFAFKNIAPSEISKKMFQNEINEKKLKLKIFKQLNSANVFNYYEKDVIIKYLKNTKFNFVNKNIPFVPSFTKLYNKIEDLRNTLKFFWSVPKDKEEKDAQIYLLKNIYYGEFLNKFVKNSKVFFKITNEVIKINKQRNQKTGHYKYQKFENIEKTVPVEYLAIIQSREMINNQDKEEKNTYIDFIQQIFLKGFIDYLNKNNLKYIESNNNNDNNDIFSKIKIKKDNKEKYDKILKNYEKHNRNKEIPHEINEFVREIKLGKILKYTENLNMFYLILKLLNHKELTNLKGSLEKYQSANKEETFSDELELINLLNLDNNRVTEDFELEANEIGKFLDFNENKIKDRKELKKFDTNKIYFDGENIIKHRAFYNIKKYGMLNLLEKIADKAKYKISLKELKEYSNKKNEIEKNYTMQQNLHRKYARPKKDEKFNDEDYKEYEKAIGNIQKYTHLKNKVEFNELNLLQGLLLKILHRLVGYTSIWERDLRFRLKGEFPENHYIEEIFNFDNSKNVKYKSGQIVEKYINFYKELYKDNVEKRSIYSDKKVKKLKQEKKDLYIRNYIAHFNYIPHAEISLLEVLENLRKLLSYDRKLKNAIMKSIVDILKEYGFVATFKIGADKKIEIQTLESEKIVHLKNLKKKKLMTDRNSEELCELVKVMFEYKALEKRPAATKKAGQAKKKKGSYPYDVPDYAYPYDVPDYAYPYDVPDYA* (SEQ ID NO: 132) c2-4 ListeriaMWISIKTLIHHLGVLFFCDYMYNRREKKIIEVKTMRITKVEV seeligeriDRKKVLISRDKNGGKLVYENEMQDNTEQIMHHKKSSFYKSVVNKTICRPEQKQMKKLVHGLLQENSQEKIKVSDVTKLNISNFLNHRFKKSLYYFPENSPDKSEEYRIEINLSQLLEDSLKKQQGTFICWESFSKDMELYINWAENYISSKTKLIKKSIRNNRIQSTESRSGQLMDRYMKDILNKNKPFDIQSVSEKYQLEKLTSALKATFKEAKKNDKEINYKLKSTLQNHERQIIEELKENSELNQFNIEIRKHLETYFPIKKTNRKVGDIRNLEIGEIQKIVNHRLKNKIVQRILQEGKLASYEIESTVNSNSLQKIKIEEAFALKFINACLFASNNLRNMVYPVCKKDILMIGEFKNSFKEIKHKKFIRQWSQFFSQEITVDDIELASWGLRGAIAPIRNEIIHLKKHSWKKFFNNPTFKVKKSKIINGKTKDVTSEFLYKETLFKDYFYSELDSVPELIINKMESSKILDYYSSDQLNQVFTIPNFELSLLTSAVPFAPSFKRVYLKGFDYQNQDEAQPDYNLKLNIYNEKAFNSEAFQAQYSLFKMVYYQVFLPQFTTNNDLFKSSVDFILTLNKERKGYAKAFQDIRKMNKDEKPSEYMSYIQSQLMLYQKKQEEKEKINHFEKFINQVFIKGFNSFIEKNRLTYICHPTKNTVPENDNIEIPFHTDMDDSNIAFWLMCKLLDAKQLSELRNEMIKFSCSLQSTEEISTFTKAREVIGLALLNGEKGCNDWKELFDDKEAWKKNMSLYVSEELLQSLPYTQEDGQTPVINRSIDLVKKYGTETILEKLFSSSDDYKVSAKDIAKLHEYDVTEKIAQQESLHKQWIEKPGLARDSAWTKKYQNVINDISNYQWAKTKVELTQVRHLHQLTIDLLSRLAGYMSIADRDFQFSSNYILERENSEYRVTSWILLSENKNKNKYNDYELYNLKNASIKVSSKNDPQLKVDLKQLRLTLEYLELFDNRLKEKRNNISHFNYLNGQLGNSILELFDDARDVLSYDRKLKNAVSKSLKEILSSHGMEVTFKPLYQTNHHLKIDKLQPKKIHHLGEKSTVSSNQVSNEYCQLVRTLLTMK (SEQ ID NO: 133) C2-17 LeptotrichiaMKVTKVGGISHKKYTSEGRLVKSESEENRTDERLSALLNMR buccalisLDMYIKNPSSTETKENQKRIGKLKKFFSNKMVYLKDNTLSL C-1013-bKNGKKENIDREYSETDILESDVRDKKNFAVLKKIYLNENVNSEELEVFRNDIKKKLNKINSLKYSFEKNKANYQKINENNIEKVEGKSKRNIIYDYYRESAKRDAYVSNVKEAFDKLYKEEDIAKLVLEIENLTKLEKYKIREFYHEIIGRKNDKENFAKIIYEEIQNVNNMKELIEKVPDMSELKKSQVFYKYYLDKEELNDKNIKYAFCHFVEIEMSQLLKNYVYKRLSNISNDKIKRIFEYQNLKKLIENKLLNKLDTYVRNCGKYNYYLQDGEIATSDFIARNRQNEAFLRNIIGVSSVAYFSLRNILETENENDITGRMRGKTVKNNKGEEKYVSGEVDKIYNENKKNEVKENLKMFYSYDFNMDNKNEIEDFFANIDEAISSIRHGIVHFNLELEGKDIFAFKNIAPSEISKKMFQNEINEKKLKLKIFRQLNSANVFRYLEKYKILNYLKRTRFEFVNKNIPFVPSFTKLYSRIDDLKNSLGIYWKTPKTNDDNKTKEIIDAQIYLLKNIYYGEFLNYFMSNNGNFFEISKEIIELNKNDKRNLKTGFYKLQKFEDIQEKIPKEYLANIQSLYMINAGNQDEEEKDTYIDFIQKIFLKGFMTYLANNGRLSLIYIGSDEETNTSLAEKKQEFDKFLKKYEQNNNIKIPYEINEFLREIKLGNILKYTERLNMFYLILKLLNHKELTNLKGSLEKYQSANKEEAFSDQLELINLLNLDNNRVTEDFELEADEIGKFLDFNGNKVKDNKELKKFDTNKIYFDGENIIKHRAFYNIKKYGMLNLLEKIADKAGYKISIEELKKYSNKKNEIEKNHKMQENLHRKYARPRKDEKFTDEDYESYKQAIENIEEYTHLKNKVEFNELNLLQGLLLRILHRLVGYTSIWERDLRFRLKGEFPENQYIEEIFNFENKKNVKYKGGQIVEKYIKFYKELHQNDEVKINKYSSANIKVLKQEKKDLYIRNYIAHFNYIPHAEISLLEVLENLRKLLSYDRKLKNAVMKSVVDILKEYGFVATFKIGADKKIGIQTLESEKIVHLKNLKKKKLMTDRNSEELCKLVKIMFEYKMEEKKSEN (SEQ ID NO: 134) C2-18 HerbinixMKLTRRRISGNSVDQKITAAFYRDMSQGLLYYDSEDNDCTD hemicellulosilyticaKVIESMDFERSWRGRILKNGEDDKNPFYMFVKGLVGSNDKIVCEPIDVDSDPDNLDILINKNLTGFGRNLKAPDSNDTLENLIRKIQAGIPEEEVLPELKKIKEMIQKDIVNRKEQLLKSIKNNRIPFSLEGSKLVPSTKKMKWLFKLIDVPNKTFNEKMLEKYWEIYDYDKLKANITNRLDKTDKKARSISRAVSEELREYHKNLRTNYNRFVSGDRPAAGLDNGGSAKYNPDKEEFLLFLKEVEQYFKKYFPVKSKHSNKSKDKSLVDKYKNYCSYKVVKKEVNRSIINQLVAGLIQQGKLLYYFYYNDTWQEDFLNSYGLSYIQVEEAFKKSVMTSLSWGINRLTSFFIDDSNTVKFDDITTKKAKEAIESNYFNKLRTCSRMQDHFKEKLAFFYPVYVKDKKDRPDDDIENLIVLVKNAIESVSYLRNRTFHFKESSLLELLKELDDKNSGQNKIDYSVAAEFIKRDIENLYDVFREQIRSLGIAEYYKADMISDCFKTCGLEFALYSPKNSLMPAFKNVYKRGANLNKAYIRDKGPKETGDQGQNSYKALEEYRELTWYIEVKNNDQSYNAYKNLLQLIYYHAFLPEVRENEALITDFINRTKEWNRKETEERLNTKNNKKHKNFDENDDITVNTYRYESIPDYQGESLDDYLKVLQRKQMARAKEVNEKEEGNNNYIQFIRDVVVWAFGAYLENKLKNYKNELQPPLSKENIGLNDTLKELFPEEKVKSPFNIKCRFSISTFIDNKGKSTDNTSAEAVKTDGKEDEKDKKNIKRKDLLCFYLFLRLLDENEICKLQHQFIKYRCSLKERRFPGNRTKLEKETELLAELEELMELVRFTMPSIPEISAKAESGYDTMIKKYFKDFIEKKVFKNPKTSNLYYHSDSKTPVTRKYMALLMRSAPLHLYKDIFKGYYLITKKECLEYIKLSNIIKDYQNSLNELHEQLERIKLKSEKQNGKDSLYLDKKDFYKVKEYVENLEQVARYKHLQHKINFESLYRIFRIHVDIAARMVGYTQDWERDMHFLFKALVYNGVLEERRFEAIFNNNDDNNDGRIVKKIQNNLNNKNRELVSMLCWNKKLNKNEFGAIIWKRNPIAHLNHFTQTEQNSKSSLESLINSLRILLAYDRKRQNAVTKTINDLLLNDYHIRIKWEGRVDEGQIYFNIKEKEDIENEPIIHLKHLHKKDCYIYKNSYMFDKQKEWICNGIKEEVYDKSILKCIGNLFKFDYEDKNKSSANPKHT (SEQ ID NO: 135) C2-19 [Eubacterium]MLRRDKEVKKLYNVFNQIQVGTKPKKWNNDEKLSPEENER rectaleRAQQKNIKMKNYKWREACSKYVESSQRIINDVIFYSYRKAKNKLRYMRKNEDILKKMQEAEKLSKFSGGKLEDFVAYTLRKSLVVSKYDTQEFDSLAAMVVFLECIGKNNISDHEREIVCKLLELIRKDFSKLDPNVKGSQGANIVRSVRNQNMIVQPQGDRFLFPQVYAKENETVTNKNVEKEGLNEFLLNYANLDDEKRAESLRKLRRILDVYFSAPNHYEKDMDITLSDNIEKEKFNVWEKHECGKKETGLFVDIPDVLMEAEAENIKLDAVVEKRERKVLNDRVRKQNIICYRYTRAVVEKYNSNEPLFFENNAINQYWIHHIENAVERILKNCKAGKLFKLRKGYLAEKVWKDAINLISIKYIALGKAVYNFALDDIWKDKKNKELGIVDERIRNGITSFDYEMIKAHENLQRELAVDIAFSVNNLARAVCDMSNLGNKESDFLLWKRNDIADKLKNKDDMASVSAVLQFFGGKSSWDINIFKDAYKGKKKYNYEVRFIDDLRKAIYCARNENFHFKTALVNDEKWNTELFGKIFERETEFCLNVEKDRFYSNNLYMFYQVSELRNMLDHLYSRSVSRAAQVPSYNSVIVRTAFPEYITNVLGYQKPSYDADTLGKWYSACYYLLKEIYYNSFLQSDRALQLFEKSVKTLSWDDKKQQRAVDNFKDHFSDIKSACTSLAQVCQIYMTEYNQQNNQIKKVRSSNDSIFDQPVYQHYKVLLKKAIANAFADYLKNNKDLFGFIGKPFKANEIREIDKEQFLPDWTSRKYEALCIEVSGSQELQKWYIVGKFLNARSLNLMVGSMRSYIQYVTDIKRRAASIGNELHVSVHDVEKVEKWVQVIEVCSLLASRTSNQFEDYFNDKDDYARYLKSYVDFSNVDMPSEYSALVDFSNEEQSDLYVDPKNPKVNRNIVHSKLFAADHILRDIVEPVSKDNIEEFYSQKAEIAYCKIKGKEITAEEQKAVLKYQKLKNRVELRDIVEYGEIINELLGQLINWSFMRERDLLYFQLGFHYDCLRNDSKKPEGYKNIKVDENSIKDAILYQIIGMYVNGVTVYAPEKDGDKLKEQCVKGGVGVKVSAFHRYSKYLGLNEKTLYNAGLEIFEVVAEHEDIINLRNGIDHFKYYLGDYRSMLSIYSEVFDRFFTYDIKYQKNVLNLLQNILLRHNVIVEPILESGFKTIGEQTKPGAKLSIRSIKSDTFQYKVKGGTLITDAKDERYLETIRKILYYAENEEDNLKKSVVVTNADKYEKNKESDDQNKQKEKKNKDNKGKKNEETKSDAEKNNNERLSYNPFANLNFKLSN (SEQ ID NO: 136) C2-20 EubacteriaceaeMKISKESHKRTAVAVMEDRVGGVVYVPGGSGIDLSNNLKK bacteriumRSMDTKSLYNVFNQIQAGTAPSEYEWKDYLSEAENKKREAQ CHKCI004KMIQKANYELRRECEDYAKKANLAVSRIIFSKKPKKIFSDDDIISHMKKQRLSKFKGRMEDFVLIALRKSLVVSTYNQEVFDSRKAATVFLKNIGKKNISADDERQIKQLMALIREDYDKWNPDKDSSDKKESSGTKVIRSIEHQNMVIQPEKNKLSLSKISNVGKKTKTKQKEKAGLDAFLKEYAQIDENSRMEYLKKLRRLLDTYFAAPSSYIKGAAVSLPENINFSSELNVWERHEAAKKVNINFVEIPESLLNAEQNNNKINKVEQEHSLEQLRTDIRRRNITCYHFANALAADERYHTLFFENMAMNQFWIHHMENAVERILKKCNVGTLFKLRIGYLSEKVWKDMLNLLSIKYIALGKAVYHFALDDIWKADIWKDASDKNSGKINDLTLKGISSFDYEMVKAQEDLQREMAVGVAFSTNNLARVTCKMDDLSDAESDFLLWNKEAIRRHVKYTEKGEILSAILQFFGGRSLWDESLFEKAYSDSNYELKFLDDLKRAIYAARNETFHFKTAAIDGGSWNTRLFGSLFEKEAGLCLNVEKNKFYSNNLVLFYKQEDLRVFLDKLYGKECSRAAQIPSYNTILPRKSFSDFMKQLLGLKEPVYGSAILDQWYSACYYLFKEVYYNLFLQDSSAKALFEKAVKALKGADKKQEKAVESFRKRYWEISKNASLAEICQSYITEYNQQNNKERKVRSANDGMFNEPIYQHYKMLLKEALKMAFASYIKNDKELKFVYKPTEKLFEVSQDNFLPNWNSEKYNTLISEVKNSPDLQKWYIVGKFMNARMLNLLLGSMRSYLQYVSDIQKRAAGLGENQLHLSAENVGQVKKWIQVLEVCLLLSVRISDKFTDYFKDEEEYASYLKEYVDFEDSAMPSDYSALLAFSNEGKIDLYVDASNPKVNRNIIQAKLYAPDMVLKKVVKKISQDECKEFNEKKEQIMQFKNKGDEVSWEEQQKILEYQKLKNRVELRDLSEYGELINELLGQLINWSYLRERDLLYFQLGFHYSCLMNESKKPDAYKTIRRGTVSIENAVLYQIIAMYINGFPVYAPEKGELKPQCKTGSAGQKIRAFCQWASMVEKKKYELYNAGLELFEVVKEHDNIIDLRNKIDHFKYYQGNDSILALYGEIFDRFFTYDMKYRNNVLNHLQNILLRHNVIIKPIISKDKKEVGRGKMKDRAAFLLEEVSSDRFTYKVKEGERKIDAKNRLYLETVRDILYFPNRAVNDKGEDVIICSKKAQDLNEKKADRDKNHDKSKDTNQKKEGKNQEEKSENKEPYSDRMTW KPFAGIKLE (SEQ ID NO: 137) C2-21Blautia sp. MKISKVDHVKSGIDQKLSSQRGMLYKQPQKKYEGKQLEEH Marseille-VRNLSRKAKALYQVFPVSGNSKMEKELQIINSFIKNILLRLDS P2398GKTSEEIVGYINTYSVASQISGDHIQELVDQHLKESLRKYTCVGDKRIYVPDIIVALLKSKFNSETLQYDNSELKILIDFIREDYLKEKQIKQIVHSIENNSTPLRIAEINGQKRLIPANVDNPKKSYIFEFLKEYAQSDPKGQESLLQHMRYLILLYLYGPDKITDDYCEEIEAWNFGSIVMDNEQLFSEEASMLIQDRIYVNQQIEEGRQSKDTAKVKKNKSKYRMLGDKIEHSINESVVKHYQEACKAVEEKDIPWIKYISDHVMSVYSSKNRVDLDKLSLPYLAKNTWNTWISFIAMKYVDMGKGVYHFAMSDVDKVGKQDNLIIGQIDPKFSDGISSFDYERIKAEDDLHRSMSGYIAFAVNNFARAICSDEFRKKNRKEDVLTVGLDEIPLYDNVKRKLLQYFGGASNWDDSIIDIIDDKDLVACIKENLYVARNVNFHFAGSEKVQKKQDDILEEIVRKETRDIGKHYRKVFYSNNVAVFYCDEDIIKLMNHLYQREKPYQAQIPSYNKVISKTYLPDLIFMLLKGKNRTKISDPSIMNMFRGTFYFLLKEIYYNDFLQASNLKEMFCEGLKNNVKNKKSEKPYQNFMRRFEELENMGMDFGEICQQIMTDYEQQNKQKKKTATAVMSEKDKKIRTLDNDTQKYKHFRTLLYIGLREAFIIYLKDEKNKEWYEFLREPVKREQPEEKEFVNKWKLNQYSDCSELILKDSLAAAWYVVAHFINQAQLNHLIGDIKNYIQFISDIDRRAKSTGNPVSESTEIQIERYRKILRVLEFAKFFCGQITNVLTDYYQDENDFSTHVGHYVKFEKKNMEPAHALQAFSNSLYACGKEKKKAGFYYDGMNPIVNRNITLASMYGNKKLLENAMNPVTEQDIRKYYSLMAELDSVLKNGAVCKSEDEQKNLRHFQNLKNRIELVDVLTLSELVNDLVAQLIGWVYIRERDMMYLQLGLHYIKLYFTDSVAEDSYLRTLDLEEGSIADGAVLYQIASLYSFNLPMYVKPNKSSVYCKKHVNSVATKFDIFEKEYCNGDETVIENGLRLFENINLHKDMVKFRDYLAHFKYFAKLDESILELYSKAYDFFFSYNIKLKKSVSYVLTNVLLSYFINAKLSFSTYKSSGNKTVQHRTTKISVVAQTDYFTYKLRSIVKNKNGVESIENDDRRCEVVNIAARDKEFVDEVCNVINYNSDK (SEQ ID NO: 138) C2-22 LeptotrichiaMGNLFGHKRWYEVRDKKDFKIKRKVKVKRNYDGNKYILNI sp. oralNENNNKEKIDNNKFIGEFVNYKKNNNVLKEFKRKFHAGNIL taxon 879FKLKGKEEIIRIENNDDFLETEEVVLYIEVYGKSEKLKALEITK str. F0557KKIIDEAIRQGITKDDKKIEIKRQENEEEIEIDIRDEYTNKTLNDCSIILRIIENDELETKKSIYEIFKNINMSLYKIIEKIIENETEKVFENRYYEEHLREKLLKDNKIDVILTNFMEIREKIKSNLEIMGFVKFYLNVSGDKKKSENKKMFVEKILNTNVDLTVEDIVDFIVKELKFWNITKRIEKVKKFNNEFLENRRNRTYIKSYVLLDKHEKFKIERENKKDKIVKFFVENIKNNSIKEKIEKILAEFKINELIKKLEKELKKGNCDTEIFGIFKKHYKVNFDSKKFSNKSDEEKELYKIIYRYLKGRIEKILVNEQKVRLKKMEKIEIEKILNESILSEKILKRVKQYTLEHIMYLGKLRHNDIVKMTVNTDDFSRLHAKEELDLELITFFASTNMELNKIFNGKEKVTDFFGFNLNGQKITLKEKVPSFKLNILKKLNFINNENNIDEKLSHFYSFQKEGYLLRNKILHNSYGNIQETKNLKGEYENVEKLIKELKVSDEEISKSLSLDVIFEGKVDIINKINSLKIGEYKDKKYLPSFSKIVLEITRKFREINKDKLFDIESEKIILNAVKYVNKILYEKITSNEENEFLKTLPDKLVKKSNNKKENKNLLSIEEYYKNAQVSSSKGDKKAIKKYQNKVTNAYLEYLENTFTEIIDFSKFNLNYDEIKTKIEERKDNKSKIIIDSISTNINITNDIEYIISIFALLNSNTYINKIRNRFFATSVWLEKQNGTKEYDYENIISILDEVLLINLLRENNITDILDLKNAIIDAKIVENDETYIKNYIFESNEEKLKKRLFCEELVDKEDIRKIFEDENFKFKSFIKKNEIGNFKINFGILSNLECNSEVEAKKIIGKNSKKLESFIQNIIDEYKSNIRTLFSSEFLEKYKEEIDNLVEDTESENKNKFEKIYYPKEHKNELYIYKKNLFLNIGNPNFDKIYGLISKDIKNVDTKILFDDDIKKNKISEIDAILKNLNDKLNGYSNDYKAKYVNKLKENDDFFAKNIQNENYSSFGEFEKDYNKVSEYKKIRDLVEFNYLNKIESYLIDINWKLAIQMARFERDMHYIVNGLRELGIIKLSGYNTGISRAYPKRNGSDGFYTTTAYYKFFDEESYKKFEKICYGFGIDLSENSEINKPENESIRNYISHFYIVRNPFADYSIAEQIDRVSNLLSYSTRYNNSTYASVFEVFKKDVNLDYDELKKKFRLIGNNDILERLMKPKKVSVLELESYNSDYIKNLIIELLTKIENTNDTL (SEQ ID NO: 139) C2-23Lachnospiraceae MKISKVDHTRMAVAKGNQHRRDEISGILYKDPTKTGSIDFDE bacteriumRFKKLNCSAKILYHVFNGIAEGSNKYKNIVDKVNNNLDRVL NK4A144FTGKSYDRKSIIDIDTVLRNVEKINAFDRISTEEREQIIDDLLEIQLRKGLRKGKAGLREVLLIGAGVIVRTDKKQEIADFLEILDEDFNKTNQAKNIKLSIENQGLVVSPVSRGEERIFDVSGAQKGKSSKKAQEKEALSAFLLDYADLDKNVRFEYLRKIRRLINLYFYVKNDDVMSLTEIPAEVNLEKDFDIWRDHEQRKEENGDFVGCPDILLADRDVKKSNSKQVKIAERQLRESIREKNIKRYRFSIKTIEKDDGTYFFANKQISVFWIHRIENAVERILGSINDKKLYRLRLGYLGEKVWKDILNFLSIKYIAVGKAVFNFAMDDLQEKDRDIEPGKISENAVNGLTSFDYEQIKADEMLQREVAVNVAFAANNLARVTVDIPQNGEKEDILLWNKSDIKKYKKNSKKGILKSILQFFGGASTWNMKMFEIAYHDQPGDYEENYLYDIIQIIYSLRNKSFHFKTYDHGDKNWNRELIGKMIEHDAERVISVEREKFHSNNLPMFYKDADLKKILDLLYSDYAGRASQVPAFNTVLVRKNFPEFLRKDMGYKVHFNNPEVENQWHSAVYYLYKEIYYNLFLRDKEVKNLFYTSLKNIRSEVSDKKQKLASDDFASRCEEIEDRSLPEICQIIMTEYNAQNFGNRKVKSQRVIEKNKDIFRHYKMLLIKTLAGAFSLYLKQERFAFIGKATPIPYETTDVKNFLPEWKSGMYASFVEEIKNNLDLQEWYIVGRFLNGRMLNQLAGSLRSYIQYAEDIERRAAENRNKLFSKPDEKIEACKKAVRVLDLCIKISTRISAEFTDYFDSEDDYADYLEKYLKYQDDAIKELSGSSYAALDHFCNKDDLKFDIYVNAGQKPILQRNIVMAKLFGPDNILSEVMEKVTESAIREYYDYLKKVSGYRVRGKCSTEKEQEDLLKFQRLKNAVEFRDVTEYAEVINELLGQLISWSYLRERDLLYFQLGFHYMCLKNKSFKPAEYVDIRRNNGTIIHNAILYQIVSMYINGLDFYSCDKEGKTLKPIETGKGVGSKIGQFIKYSQYLYNDPSYKLEIYNAGLEVFENIDEHDNITDLRKYVDHFKYYAYGNKMSLLDLYSEFFDRFFTYDMKYQKNVVNVLENILLRHFVIFYPKFGSGKKDVGIRDCKKERAQIEISEQSLTSEDFMFKLDDKAGEEAKKFPARDERYLQTIAKLLYYPNEIEDMNRFMKK GETINKKVQFNRKKKITRKQKNNSSNEVLSSTMGYLFKNIKL (SEQ ID NO: 140) C2-24 ChloroflexusMTDQVRREEVAAGELADTPLAAAQTPAADAAVAATPAPAE aggregansAVAPTPEQAVDQPATTGESEAPVTTAQAAAHEAEPAEATGASFTPVSEQQPQKPRRLKDLQPGMELEGKVTSIALYGIFVDVGVGRDGLVHISEMSDRRIDTPSELVQIGDTVKVWVKSVDLDARRISLTMLNPSRGEKPRRSRQSQPAQPQPRRQEVDREKLASLKVGEIVEGVITGFAPFGAFADIGVGKDGLIHISELSEGRVEKPEDAVKVGERYQFKVLEIDGEGTRISLSLRRAQRTQRMQQLEPGQIIEGTVSGIATFGAFVDIGVGRDGLVHISALAPHRVAKVEDVVKVGDKVKVKVLGVDPQSKRISLTMRLEEEQPATTAGDEAAEPAEEVTPTRRGNLERFAAAAQTARERSERGERSERGERRERRERRPAQSSPDTYIVGEDDDESFEGNATIEDLLTKFGGSSSRRDRDRRRRHEDDDDEEMERPSNRRQREAIRRTLQQIGYDE (SEQ ID NO: 141) C2-25Demequina MDLTWHALLILFIVALLAGELDTLAGGGGLLTVPALLLTGIP aurantiacaPLQALGTNKLQSSFGTGMATYQVIRKKRVHWRDVRWPMVWAFLGSAAGAVAVQFIDTDALLIIIPVVLALVAAYFLFVPKSHLPPPEPRMSDPAYEATLVPIIGAYDGAFGPGTGSLYALSGVALRAKTLVQSTAIAKTLNFATNFAALLVFAFAGHMLWTVGAVMIAGQLIGAYAGSHMLFRVNPLVLRVLIVVMSLGMLIRVL LD (SEQ ID NO: 142) C2-26Thalassospira MRIIKPYGRSHVEGVATQEPRRKLRLNSSPDISRDIPGFAQSH sp.DALIIAQWISAIDKIATKPKPDKKPTQAQINLRTTLGDAAWQ TSL5-1HVMAENLLPAATDPAIREKLHLIWQSKIAPWGTARPQAEKDGKPTPKGGWYERFCGVLSPEAITQNVARQIAKDIYDHLHVAAKRKGREPAKQGESSNKPGKFKPDRKRGLIEERAESIAKNALRPGSHAPCPWGPDDQATYEQAGDVAGQIYAAARDCLEEKKRRSGNRNTSSVQYLPRDLAAKILYAQYGRVFGPDTTIKAALDEQPSLFALHKAIKDCYHRLINDARKRDILRILPRNMAALFRLVRAQYDNRDINALIRLGKVIHYHASEQGKSEHHGIRDYWPSQQDIQNSRFWGSDGQADIKRHEAFSRIWRHIIALASRTLHDWADPHSQKFSGENDDILLLAKDAIEDDVFKAGHYERKCDVLFGAQASLFCGAEDFEKAILKQAITGTGNLRNATFHFKGKVRFEKELQELTKDVPVEVQSAIAALWQKDAEGRTRQIAETLQAVLAGHFLTEEQNRHIFAALTAAMAQPGDVPLPRLRRVLARHDSICQRGRILPLSPCPDRAKLEESPALTCQYTVLKMLYDGPFRAWLAQQNSTILNHYIDSTIARTDKAARDMNGRKLAQAEKDLITSRAADLPRLSVDEKMGDFLARLTAATATEMRVQRGYQSDGENAQKQAAFIGQFECDVIGRAFADFLNQSGFDFVLKLKADTPQPDAAQCDVTALIAPDDISVSPPQAWQQVLYFILHLVPVDDASHLLHQIRKWQVLEGKEKPAQIAHDVQSVLMLYLDMHDAKFTGGAALHGIEKFAEFFAHAADFRAVFPPQSLQDQDRSIPRRGLREIVRFGHLPLLQHMSGTVQITHDNVVAWQAARTAGATGMSPIARRQKQREELHALAVERTARFRNADLQNYMHALVDVIKHRQLSAQVTLSDQVRLHRLMMGVLGRLVDYAGLWERDLYFVVLALLYHHGATPDDVFKGQGKKNLADGQVVAALKPKNRKAAAPVGVFDDLDHYGIYQDDRQSIRNGLSHFNMLRGGKAPDLSHWVNQTRSLVAHDRKLKNAVAKSVIEMLAREGFDLDWGIQTDRGQHILSHGKIRTRQAQHFQKSRLHIVKKSAKPDKNDTVKIRENLHGDAMVERVVQLFAAQVQKRYDITVEKRLDHLFLKPQDQKGKNGIHTHNGWSKTEKKRRPSRENRKGNH EN (SEQ ID NO: 143) C2-27SAMN044 MKFSKESHRKTAVGVTESNGIIGLLYKDPLNEKEKIEDVVNQ 87830_13920RANSTKRLFNLFGTEATSKDISRASKDLAKVVNKAIGNLKGN [PseudobutyrivibrioKKFNKKEQITKGLNTKIIVEELKNVLKDEKKLIVNKDIIDEAC sp. OR37]SRLLKTSFRTAKTKQAVKMILTAVLIENTNLSKEDEAFVHEYFVKKLVNEYNKTSVKKQIPVALSNQNMVIQPNSVNGTLEISETKKSKETKTTEKDAFRAFLRDYATLDENRRHKMRLCLRNLVNLYFYGETSVSKDDFDEWRDHEDKKQNDELFVKKIVSIKTDRKGNVKEVLDVDATIDAIRTNNIACYRRALAYANENPDVFFSDTMLNKFWIHHVENEVERIYGHINNNTGDYKYQLGYLSEKVWKGIINYLSIKYIAEGKAVYNYAMNALAKDNNSNAFGKLDEKFVNGITSFEYERIKAEETLQRECAVNIAFAANHLANATVDLNEKDSDFLLLKHEDNKDTLGAVARPNILRNILQFFGGKSRWNDFDFSGIDEIQLLDDLRKMIYSLRNSSFHFKTENIDNDSWNTKLIGDMFAYDFNMAGNVQKDKMYSNNVPMFYSTSDIEKMLDRLYAEVHERASQVPSFNSVFVRKNFPDYLKNDLKITSAFGVDDALKWQSAVYYVCKEIYYNDFLQNPETFTMLKDYVQCLPIDIDKSMDQKLKSERNAHKNFKEAFATYCKECDSLSAICQMIMTEYNNQNKGNRKVISARTKDGDKLIYKHYKMILFEALKNVFTIYLEKNINTYGFLKKPKLINNVPAIEEFLPNYNGRQYETLVNRITEETELQKWYIVGRLLNPKQVNQLIGNERSYVQYVNDVARRAKQTGNNLSNDNIAWDVKNIIQIFDVCTKLNGVTSNILEDYFDDGDDYARYLKNFVDYTNKNNDHSATLLGDFCAKEIDGIKIGIYHDGTNPIVNRNIIQCKLYGATGIISDLTKDGSILSVDYEIIKKYMQMQKEIKVYQQKGICKTKEEQQNLKKYQELKNIVELRNIIDYSEILDELQGQLINWGYLRERDLMYFQLGFHYLCLHNESKKPVGYNNAGDISGAVLYQIVAMYTNGLSLIDANGKSKKNAKASAGAKVGSFCSYSKEIRGVDKDTKEDDDPIYLAGVELFENINEHQQCINLRNYIEHFHYYAKHDRSMLDLYSEVFDRFFTYDMKYTKNVPNMMYNILLQHLVVPAFEFGSSEKRLDDNDEQTKPRAMFTLREKNGLSSEQFTYRLGDGNSTVKLSARGDDYLRAVASLLYYPDRAPEGLIRDAEAEDKFAKINHSNPKSDNRNNRGNFKNPKVQWYNNKTKRK (SEQ ID NO: 144) C2-28 SAMN029MKISKVDHRKTAVKITDNKGAEGFIYQDPTRDSSTMEQIISN 10398_00008RARSSKVLFNIFGDTKKSKDLNKYTESLIIYVNKAIKSLKGDK [ButyrivibrioRNNKYEEITESLKTERVLNALIQAGNEFTCSENNIEDALNKY sp.LKKSFRVGNTKSALKKLLMAAYCGYKLSIEEKEEIQNYFVD YAB3001]KLVKEYNKDTVLKYTAKSLKHQNMVVQPDTDNHVFLPSRIAGATQNKMSEKEALTEFLKAYAVLDEEKRHNLRIILRKLVNLYFYESPDFIYPENNEWKEHDDRKNKTETFVSPVKVNEEKNGKTFVKIDVPATKDLIRLKNIECYRRSVAETAGNPITYFTDHNISKFWIHHIENEVEKIFALLKSNWKDYQFSVGYISEKVWKEIINYLSIKYIAIGKAVYNYALEDIKKNDGTLNFGVIDPSFYDGINSFEYEKIKAEETFQREVAVYVSFAVNHLSSATVKLSEAQSDMLVLNKNDIEKIAYGNTKRNILQFFGGQSKWKEFDFDRYINPVNYTDIDFLFDIKKMVYSLRNESFHFTTTDTESDWNKNLISAMFEYECRRISTVQKNKFFSNNLPLFYGENSLERVLHKLYDDYVDRMSQVPSFGNVFVRKKFPDYMKEIGIKHNLSSEDNLKLQGALYFLYKEIYYNAFISSEKAMKIFVDLVNKLDTNARDDKGRITHEAMAHKNFKDAISHYMTHDCSLADICQKIMTEYNQQNTGHRKKQTTYSSEKNPEIFRHYKMILFMLLQKAMTEYISSEEIFDFIMKPNSPKTDIKEEEFLPQYKSCAYDNLIKLIADNVELQKWYITARLLSPREVNQLIGSFRSYKQFVSDIERRAKETNNSLSKSGMTVDVENITKVLDLCTKLNGRFSNELTDYFDSKDDYAVYVSKFLDFGFKIDEKFPAALLGEFCNKEENGKKIGIYHNGTEPILNSNIIKSKLYGITDVVSRAVKPVSEKLIREYLQQEVKIKPYLENGVCKNKEEQAALRKYQELKNRIEFRDIVEYSEIINELMGQLINFSYLRERDLMYFQLGFHYLCLNNYGAKPEGYYSIVNDKRTIKGAILYQIVAMYTYGLPIYHYVDGTISDRRKNKKTVLDTLNSSETVGAKIKYFIYYSDELFNDSLILYNAGLELFENINEHENIVNLRKYIDHFKYYVSQDRSLLDIYSEVFDRYFTYDRKYKKNVMNLFSNIMLKHFIITDFEFSTGEKTIGEKNTAKKECAKVRIKRGGLSSDKFTYKFKDAKPIELSAKNTEFLDGVARILYYPENVVLTDLVRNSEVEDEKRIEKYDRNHNSSPTRKDKTYKQDVKKNYNKKTSKAFDSSKLDTKSVGNNLSDNPVLKQFLSESKKK R (SEQ ID NO: 145) C2-29Blautia sp. MKISKVDHVKSGIDQKLSSQRGMLYKQPQKKYEGKQLEEH Marseille-VRNLSRKAKALYQVFPVSGNSKMEKELQIINSFIKNILLRLDS P2398GKTSEEIVGYINTYSVASQISGDHIQELVDQHLKESLRKYTCVGDKRIYVPDIIVALLKSKFNSETLQYDNSELKILIDFIREDYLKEKQIKQIVHSIENNSTPLRIAEINGQKRLIPANVDNPKKSYIFEFLKEYAQSDPKGQESLLQHMRYLILLYLYGPDKITDDYCEEIEAWNFGSIVMDNEQLFSEEASMLIQDRIYVNQQIEEGRQSKDTAKVKKNKSKYRMLGDKIEHSINESVVKHYQEACKAVEEKDIPWIKYISDHVMSVYSSKNRVDLDKLSLPYLAKNTWNTWISFIAMKYVDMGKGVYHFAMSDVDKVGKQDNLIIGQIDPKFSDGISSFDYERIKAEDDLHRSMSGYIAFAVNNFARAICSDEFRKKNRKEDVLTVGLDEIPLYDNVKRKLLQYFGGASNWDDSIIDIIDDKDLVACIKENLYVARNVNFHFAGSEKVQKKQDDILEEIVRKETRDIGKHYRKVFYSNNVAVFYCDEDIIKLMNHLYQREKPYQAQIPSYNKVISKTYLPDLIFMLLKGKNRTKISDPSIMNMFRGTFYFLLKEIYYNDFLQASNLKEMFCEGLKNNVKNKKSEKPYQNFMRRFEELENMGMDFGEICQQIMTDYEQQNKQKKKTATAVMSEKDKKIRTLDNDTQKYKHFRTLLYIGLREAFIIYLKDEKNKEWYEFLREPVKREQPEEKEFVNKWKLNQYSDCSELILKDSLAAAWYVVAHFINQAQLNHLIGDIKNYIQFISDIDRRAKSTGNPVSESTEIQIERYRKILRVLEFAKFFCGQITNVLTDYYQDENDFSTHVGHYVKFEKKNMEPAHALQAFSNSLYACGKEKKKAGFYYDGMNPIVNRNITLASMYGNKKLLENAMNPVTEQDIRKYYSLMAELDSVLKNGAVCKSEDEQKNLRHFQNLKNRIELVDVLTLSELVNDLVAQLIGWVYIRERDMMYLQLGLHYIKLYFTDSVAEDSYLRTLDLEEGSIADGAVLYQIASLYSFNLPMYVKPNKSSVYCKKHVNSVATKFDIFEKEYCNGDETVIENGLRLFENINLHKDMVKFRDYLAHFKYFAKLDESILELYSKAYDFFFSYNIKLKKSVSYVLTNVLLSYFINAKLSFSTYKSSGNKTVQHRTTKISVVAQTDYFTYKLRSIVKNKNGVESIENDDRRCEVVNIAARDKEFVDEVCNVINYNSDK (SEQ ID NO: 146) C2-30 LeptotrichiaMKITKIDGISHKKYIKEGKLVKSTSEENKTDERLSELLTIRLD sp.TYIKNPDNASEEENRIRRENLKEFFSNKVLYLKDGILYLKDR Marseille-REKNQLQNKNYSEEDISEYDLKNKNNFLVLKKILLNEDINSE P3007ELEIFRNDFEKKLDKINSLKYSLEENKANYQKINENNIKKVEGKSKRNIFYNYYKDSAKRNDYINNIQEAFDKLYKKEDIENLFFLIENSKKHEKYKIRECYHKIIGRKNDKENFATIIYEEIQNVNNMKELIEKVPNVSELKKSQVFYKYYLNKEKLNDENIKYVFCHFVEIEMSKLLKNYVYKKPSNISNDKVKRIFEYQSLKKLIENKLLNKLDTYVRNCGKYSFYLQDGEIATSDFIVGNRQNEAFLRNIIGVSSTAYFSLRNILETENENDITGRMRGKTVKNNKGEEKYISGEIDKLYDNNKQNEVKKNLKMFYSYDFNMNSKKEIEDFFSNIDEAISSIRHGIVHFNLELEGKDIFTFKNIVPSQISKKMFHDEINEKKLKLKIFKQLNSANVFRYLEKYKILNYLNRTRFEFVNKNIPFVPSFTKLYSRIDDLKNSLGIYWKTPKTNDDNKTKEITDAQIYLLKNIYYGEFLNYFMSNNGNFFEITKEIIELNKNDKRNLKTGFYKLQKFENLQEKTPKEYLANIQSLYMINAGNQDEEEKDTYIDFIQKIFLKGFMTYLANNGRLSLIYIGSDEETNTSLAEKKQEFDKFLKKYEQNNNIEIPYEINEFVREIKLGKILKYTERLNMFYLILKLLNHKELTNLKGSLEKYQSANKEEAFSDQLELINLLNLDNNRVTEDFELEADEIGKFLDFNGNKVKDNKELKKFDTNKIYFDGENIIKHRAFYNIKKYGMLNLLEKISDEAKYKISIEELKNYSKKKNEIEENHTTQENLHRKYARPRKDEKFTDEDYKKYEKAIRNIQQYTHLKNKVEFNELNLLQSLLLRILHRLVGYTSIWERDLRFRLKGEFPENQYIEEIFNFDNSKNVKYKNGQIVEKYINFYKELYKDDTEKISIYSDKKVKELKKEKKDLYIRNYIAHFNYIPNAEISLLEMLENLRKLLSYDRKLKNAIMKSIVDILKEYGFVVTFKIEKDKKIRIESLKSEEVVHLKKLKLKDNDKKKEPIKTYRNSKELCKLVKVMFEYKMKEKKSEN (SEQ ID NO: 147) C2-31 BacteroidesMRITKVKVKESSDQKDKMVLIHRKVGEGTLVLDENLADLTA ihuaePIIDKYKDKSFELSLLKQTLVSEKEMNIPKCDKCTAKERCLSCKQREKRLKEVRGAIEKTIGAVIAGRDIIPRLNIFNEDEICWLIKPKLRNEFTFKDVNKQVVKLNLPKVLVEYSKKNDPTLFLAYQQWIAAYLKNKKGHIKKSILNNRVVIDYSDESKLSKRKQALELWGEEYETNQRIALESYHTSYNIGELVTLLPNPEEYVSDKGEIRPAFHYKLKNVLQMHQSTVFGTNEILCINPIFNENRANIQLSAYNLEVVKYFEHYFPIKKKKKNLSLNQAIYYLKVETLKERLSLQLENALRMNLLQKGKIKKHEFDKNTCSNTLSQIKRDEFFVLNLVEMCAFAANNIRNIVDKEQVNEILSKKDLCNSLSKNTIDKELCTKFYGADFSQIPVAIWAMRGSVQQIRNEIVHYKAEAIDKIFALKTFEYDDMEKDYSDTPFKQYLELSIEKIDSFFIEQLSSNDVLNYYCTEDVNKLLNKCKLSLRRTSIPFAPGFKTIYELGCHLQDSSNTYRIGHYLMLIGGRVANSTVTKASKAYPAYRFMLKLIYNHLFLNKFLDNHNKRFFMKAVAFVLKDNRENARNKFQYAFKEIRMMNNDESIASYMSYIHSLSVQEQEKKGDKNDKVRYNTEKFIEKVFVKGFDDFLSWLGVEFILSPNQEERDKTVTREEYENLMIKDRVEHSINSNQESHIAFFTFCKLLDANHLSDLRNEWIKFRSSGDKEGFSYNFAIDIIELCLLTVDRVEQRRDGYKEQTELKEYLSFFIKGNESENTVWKGFYFQQDNYTPVLYSPIELIRKYGTLELLKLIIVDEDKITQGEFEEWQTLKKVVEDKVTRRNELHQEWEDMKNKSSFSQEKCSIYQKLCRDIDRYNWLDNKLHLVHLRKLHNLVIQILSRMARFIALWDRDFVLLDASRANDDYKLLSFFNFRDFINAKKTKTDDELLAEFGSKIEKKNAPFIKAEDVPLMVECIEAKRSFYQKVFFRNNLQVLADRNFIAHYNYISKTAKCSLFEMIIKLRTLMYYDRKLRNAVVKSIANVFDQNGMVLQLSLDDSHELKVDKVISKRIVHLKNNNIMTDQVPEEYYKICRRL LEMKK (SEQ ID NO: 148) C2-32SAMN052 MEFRDSIFKSLLQKEIEKAPLCFAEKLISGGVFSYYPSERLKEF 16357_1045VGNHPFSLFRKTMPFSPGFKRVMKSGGNYQNANRDGRFYD [PorphyromonadaceaeLDIGVYLPKDGFGDEEWNARYFLMKLIYNQLFLPYFADAEN bacteriumHLFRECVDFVKRVNRDYNCKNNNSEEQAFIDIRSMREDESIA KH3CP3RA]DYLAFIQSNIIIEENKKKETNKEGQINFNKFLLQVFVKGFDSFLKDRTELNFLQLPELQGDGTRGDDLESLDKLGAVVAVDLKLDATGIDADLNENISFYTFCKLLDSNHLSRLRNEIIKYQSANSDFSHNEDFDYDRIISIIELCMLSADHVSTNDNESIFPNNDKDFSGIRPYLSTDAKVETFEDLYVHSDAKTPITNATMVLNWKYGTDKLFERLMISDQDFLVTEKDYFVWKELKKDIEEKIKLREELHSLWVNTPKGKKGAKKKNGRETTGEFSEENKKEYLEVCREIDRYVNLDNKLHFVHLKRMHSLLIELLGRFVGFTYLFERDYQYYHLEIRSRRNKDAGVVDKLEYNKIKDQNKYDKDDFFACTFLYEKANKVRNFIAHFNYLTMWNSPQEEEHNSNLSGAKNSSGRQNLKCSLTELINELREVMSYDRKLKNAVTKAVIDLFDKHGMVIKFRIVNNNNNDNKNKHHLELDDIVPKKIMHLRGIKLKRQDGKPIPIQTDSVDPLYCRMWKKLLDLKPTPF (SEQ ID NO: 149) C2-33 ListeriaMHDAWAENPKKPQSDAFLKEYKACCEAIDTYNWHKNKAT ripariaLVYVNELHHLLIDILGRLVGYVAIADRDFQCMANQYLKSSGHTERVDSWINTIRKNRPDYIEKLDIFMNKAGLFVSEKNGRNYIAHLNYLSPKHKYSLLYLFEKLREMLKYDRKLKNAVTKSLIDLLDKHGMCVVFANLKNNKHRLVIASLKPKKIETFKWKKIK (SEQ ID NO: 150) C2-34Insolitispirillum MRIIRPYGSSTVASPSPQDAQPLRSLQRQNGTFDVAEFSRRHP peregrinumELVLAQWVAMLDKIIRKPAPGKNSTALPRPTAEQRRLRQQVGAALWAEMQRHTPVPPELKAVWDSKVHPYSKDNAPATAKTPSHRGRWYDRFGDPETSAATVAEGVRRHLLDSAQPFRANGGQPKGKGVIEHRALTIQNGTLLHHHQSEKAGPLPEDWSTYRADELVSTIGKDARWIKVAASLYQHYGRIFGPTTPISEAQTRPEFVLHTAVKAYYRRLFKERKLPAERLERLLPRTGEALRHAVTVQHGNRSLADAVRIGKILHYGWLQNGEPDPWPDDAALYSSRYWGSDGQTDIKHSEAVSRVWRRALTAAQRTLTSWLYPAGTDAGDILLIGQKPDSIDRNRLPLLYGDSTRHWTRSPGDVWLFLKQTLENLRNSSFHFKTLSAFTSHLDGTCESEPAEQQAAQALWQDDRQQDHQQVFLSLRALDATTYLPTGPLHRIVNAVQSTDATLPLPRFRRVVTRAANTRLKGFPVEPVNRRTMEDDPLLRCRYGVLKLLYERGFRAWLETRPSIASCLDQSLKRSTKAAQTINGKNSPQGVEILSRATKLLQAEGGGGHGIHDLFDRLYAATAREMRVQVGYHHDAEAARQQAEFIEDLKCEVVARAFCAYLKTLGIQGDTFRRQPEPLPTWPDLPDLPSSTIGTAQAALYSVLHLMPVEDVGSLLHQLRRWLVALQARGGEDGTAITATIPLLELYLNRHDAKFSGGGAGTGLRWDDWQVFFDCQATFDRVFPPGPALDSHRLPLRGLREVLRFGRVNDLAALIGQDKITAAEVDRWHTAEQTIAAQQQRREALHEQLSRKKGTDAEVDEYRALVTAIADHRHLTAHVTLSNVVRLHRLMTTVLGRLVDYGGLWERDLTFVTLYEAHRLGGLRNLLSESRVNKFLDGQTPAALSKKNNAEENGMISKVLGDKARRQIRNDFAHFNMLQQGKKTINLTDEINNARKLMAHDRKLKNAITRSVTTLLQQDGLDIVWTMDASHRLTDAKIDSRNAIHLHKTHNRANIREPLHGKSYCRWVAALFGATSTPSATKKSDKIR (SEQ ID NO: 151)

Example 3: Characterization of Cas13b Orthologs

The PFS motif was determined for the different Cas13b orthologs asdescribed in Smargon et al. 2016 and as illustrated in FIG. 6 . Heatmapsof the ratio of safely depleted (>5σ above mean depletion ofnon-targeting spacers) spacers to all spacers in the screen weregenerated. For some orthologs, experiments were repeated in the presenceof the accessory protein csx27 or csx28 The results are provided inFIGS. 7-10 . The following PFS were derived for the different orthologs:

TABLE 4 Ortholog No. Accessory protein 5′PFS 3′PFS Bergeyella zoohelcum1 A NGA Bergeyella zoohelcum 1 csx27 A NGA Prevotella intermedia 2 A NGAPrevotella intermedia 2 csx28 A NGA Prevotella buccae 3 A NGA Prevotellabuccae 3 csx28 A NGA Bacteroides pyogenes 5 A NGA Alistipes sp. ZOR00096 TG NA(G) Prevotella sp. MA2016 7 AT Riemerella anatipestifer 8 A NGARiemerella anatipestifer 8 csx28 A NGA Prevotella aurantiaca 9 G NAAPrevotella aurantiaca 9 csx28 Prevotella saccharolytica 10 AG Prevotellaintermedia 12 AG Capnocytophaga canimorsus 13 A NAA Capnocytophagacanimorsus 13 csx27 A NAA Porphyromonas gulae 14 A NAA Porphyromonasgulae 14 csx28 A NGA Prevotella sp. P5-125 15 AT Flavobacteriumbranchiophilum 16 TG Flavobacterium branchiophilum 16 csx27 TA Myroidesodoratimimus 17 T NAA Porphyromonas gingivalis 19 A NAA Porphyromonasgingivalis 19 csx28 A Prevotella intermedia 21 A NGA Prevotellaintermedia 21 csx28 A NGA

Example 4: Collateral Activity of Cas13b Orthologs

In order to determine the collateral activity of Cas13b orthologs ineukaryotic cells, experiments with target specific guides in eukaryoticcells were carried out in the presence of a G-luciferase r C-luciferasereporter construct. The results are provided in FIGS. 17A-G. Collateralactivity in vivo was effectively observed for different Cas13borthologs.

Example 5: Identification of Further Cas13b Orthologs

Further Cas13b orthologs can be identified using a biocomputationalpipeline relying on either proximity to CRISPR associated proteins orCRISPR arrays per se. An exemplary method may include downloading of allprokaryotic genomes (from the NCBI database), running an algorithm onthese genomes to identify CRISPR arrays, storing proteins clusteredwithin 10 kb of the identified array as as putative CRISPR systems andfurther selecting candidate systems based on the absence of proteinsgreater than 700 amino acids in size. Further selection criteria can bebased on the presence of only one effector protein between 900-1800amino acids in size and based on functionality and compatibilityconsiderations.

Example 6

Efficient and precise nucleic acid editing holds great promise fortreating genetic disease, particularly at the level of RNA, wheredisease-relevant transcripts can be rescued to yield functional proteinproducts. Type VI CRISPR-Cas systems contain the programmablesingle-effector RNA-guided RNases Cas13. Here, we profile the diversityof Type VI systems to engineer a Cas13 ortholog capable of robustknockdown and demonstrate RNA editing by using catalytically-inactiveCas13 (dCas13) to direct adenosine deaminase activity to transcripts inmammalian cells. By fusing the ADAR2 deaminase domain to dCas13 andengineering guide RNAs to create an optimal RNA duplex substrate, weachieve targeted editing of specific single adenosines to inosines(which is read out as guanosine during translation) with efficienciesroutinely ranging from 20-40% and up to 89%. This system, referred to asRNA Editing for Programmable A to I Replacement (REPAIR), can be furtherengineered to achieve high specificity. An engineered variant, REPAIRv2,displays greater than 170-fold increase in specificity while maintainingrobust on-target A to I editing. We use REPAIRv2 to edit full-lengthtranscripts containing known pathogenic mutations and create functionaltruncated versions suitable for packaging in adeno-associated viral(AAV) vectors. REPAIR presents a promising RNA editing platform withbroad applicability for research, therapeutics, and biotechnology.Precise nucleic acid editing technologies are valuable for studyingcellular function and as novel therapeutics. Although current editingtools, such as the Cas9 nuclease, can achieve programmable modificationof genomic loci, edits are often heterogenous due to insertions ordeletions or require a donor template for precise editing. Base editors,such as dCas9-APOBEC fusions, allow for editing without generating adouble stranded break, but may lack precision due to the nature ofcytidine deaminase activity, which edits any cytidine in a targetwindow. Furthermore, the requirement for a protospacer adjacent motif(PAM) limits the number of possible editing sites. Here, we describe thedevelopment of a precise and flexible RNA base editing tool using theRNA-guided RNA targeting Cas13 enzyme from type VI prokaryotic clusteredregularly interspaced short palindromic repeats (CRISPR) adaptive immunesystem.

Precise nucleic acid editing technologies are valuable for studyingcellular function and as novel therapeutics. Current editing tools,based on programmable nucleases such as the prokaryotic clusteredregularly interspaced short palindromic repeats (CRISPR)-associatednucleases Cas9 (1-4) or Cpf1(5), have been widely adopted for mediatingtargeted DNA cleavage which in turn drives targeted gene disruptionthrough non-homologous end joining (NHEJ) or precise gene editingthrough template-dependent homology-directed repair (HDR)(6). NHEJutilizes host machineries that are active in both dividing andpost-mitotic cells and provides efficient gene disruption by generatinga mixture of insertion or deletion (indel) mutations that can lead toframe shifts in protein coding genes. HDR, in contrast, is mediated byhost machineries whose expression is largely limited to replicatingcells. As such, the development of gene-editing capabilities inpost-mitotic cells remains a major challenge. Recently, DNA baseeditors, such as the use of catalytically inactive Cas9 (dCas9) totarget cytidine deaminase activity to specific genome targets to effectcytosine to thymine conversions within a target window, allow forediting without generating a DNA double strand break and significantlyreduces the formation of indels(7, 8). However the targeting range ofDNA base editors is limited due to the requirement of Cas9 for aprotospacer adjacent motif (PAM) at the editing site(9). Here, wedescribe the development of a precise and flexible RNA base editingtechnology using the type VI CRISPR-associated RNA-guided RNaseCas13(10-13).

Cas13 enzymes have two Higher Eukaryotes and ProkaryotesNucleotide-binding (HEPN) endoRNase domains that mediate precise RNAcleavage(10, 11). Three Cas13 protein families have been identified todate: Cas13a (previously known as C2c2), Cas13b, and Cas13c(12, 13). Werecently reported Cas13a enzymes can be adapted as tools for nucleicacid detection(14) as well as mammalian and plant cell RNA knockdown andtranscript tracking(15). The RNA-guided nature of Cas13 enzymes makesthem attractive tool for RNA binding and perturbation applications.

The adenosine deaminase acting on RNA (ADAR) family of enzymes mediatesendogenous editing of transcripts via hydrolytic deamination ofadenosine to inosine, a nucleobase that is functionally equivalent toguanosine in translation and splicing(16). There are two functionalhuman ADAR orthologs, ADAR1 and ADAR2, which consist of N-terminaldouble stranded RNA-binding domains and a C-terminal catalyticdeamination domain. Endogenous target sites of ADAR1 and ADAR2 containsubstantial double stranded identity, and the catalytic domains requireduplexed regions for efficient editing in vitro and in vivo(17, 18).Although ADAR proteins have preferred motifs for editing that couldrestrict the potential flexibility of targeting, hyperactive mutants,such as ADAR(E488Q)(19), relax sequence constraints and improveadenosine to inosine editing rates. ADARs preferentially deaminateadenosines opposite cytidine bases in RNA duplexes(20), providing apromising opportunity for precise base editing. Although previousapproaches have engineered targeted ADAR fusions via RNA guides (21-24),the specificity of these approaches has not been reported and theirrespective targeting mechanisms rely on RNA-RNA hybridization withoutthe assistance of protein partners that may enhance target recognitionand stringency.

Here we assay the entire family of Cas13 enzymes for RNA knockdownactivity in mammalian cells and identify the Cas13b ortholog fromPrevotella sp. P5-125 (PspCas13b) as the most efficient and specific formammalian cell applications. We then fuse the ADAR2 deaminase domain(ADARDD) to catalytically inactive PspCas13b and demonstrate RNA editingfor programmable A to I (G) replacement (REPAIR) of reporter andendogenous transcripts as well as disease-relevant mutations. Lastly, weemploy a rational mutagenesis scheme to improve the specificity ofdCas13b-ADAR2DD fusions to generate REPAIRv2 with more than 170 foldincrease in specificity.

Methods

Design and Cloning of Bacterial Constructs

Mammalian codon optimized Cas13b constructs were cloned into thechloramphenicol resistant pACYC184 vector under control of the Lacpromoter. Two corresponding direct-repeat (DR) sequences separated byBsaI restriction sites were then inserted downstream of Cas13b, undercontrol of the pJ23119 promoter. Last, oligos for targeting spacers werephosphorylated using T4 PNK (New England Biolabs), annealed and ligatedinto BsaI digested vectors using T7 ligase (Enzymatics) to generatetargeting Cas13b vectors.

Bacterial PFS Screens

Ampicillin resistance plasmids for PFS screens were cloned by insertingPCR products containing Cas13b targets with 2 5′ randomized nucleotidesand 4 3′ randomized nucleotides separated by a target site immediatelydownstream of the start codon of the ampicillin resistance gene blausing NEB Gibson Assembly (New England Biolabs). 100 ng ofampicillin-resistant target plasmids were then electroporated with65-100 ng chloramphenicol-resistant Cas13b bacterial targeting plasmidsinto Endura Electrocompetent Cells. Plasmids were added to cells,incubated 15 minutes on ice, electroporated using the manufacturer'sprotocol, and then 950 uL of recovery media was added to cells before aone hour outgrowth at 37C. The outgrowth was plated onto chloramphenicoland ampicillin double selection plates. Serial dilutions of theoutgrowth were used to estimate the cfu/ng DNA. 16 hours post plating,cells were scraped off plates and surviving plasmid DNA harvested usingthe Qiagen Plasmid Plus Maxi Kit (Qiagen). Surviving Cas13b targetsequences and their flanking regions were amplified by PCR and sequencedusing an Illumina NextSeq. To assess PFS preferences, the positionscontaining randomized nucleotides in the original library wereextracted, and sequences depleted relative to the vector only conditionthat were present in both bioreplicates were extracted using custompython scripts. The −log 2 of the ratio of PFS abundance in the Cas13bcondition compared to the vector only control was then used to calculatepreferred motifs. Specifically, all sequences having −log2(sample/vector) depletion ratios above a specific threshold were usedto generate weblogos of sequence motifs (weblogo.berkeley.edu). Thespecific depletion ratio values used to generate weblogos for eachCas13b ortholog are listed in Table 9.

Design and Cloning of Mammalian Constructs for RNA Interference

To generate vectors for testing Cas13 orthologs in mammalian cells,mammalian codon optimized Cas13a, Cas13b, and Cas13c genes were PCRamplified and golden-gate cloned into a mammalian expression vectorcontaining dual NLS sequences and a C-terminal msfGFP, under control ofthe EF1alpha promoter. For further optimization Cas13 orthologs weregolden gate cloned into destination vectors containing differentC-terminal localization tags under control of the EF1alpha promoter.

The dual luciferase reporter was cloned by PCR amplifying Gaussia andCypridina luciferase coding DNA, the EF1alpha and CMV promoters andassembly using the NEB Gibson Assembly (New England Biolabs).

For expression of mammalian guide RNA for Cas13a, Cas13b, or Cas13corthologs, the corresponding direct repeat sequences were synthesizedwith golden-gate acceptor sites and cloned under U6 expression viarestriction digest cloning. Individual guides were then cloned into thecorresponding expression backbones for each ortholog by golden gatecloning.

Cloning of Pooled Mismatch Libraries for Cas13 Interference Specificity

Pooled mismatch library target sites were created by PCR. Oligoscontaining semi-degenerate target sequences in G-luciferase containing amixture of 94% of the correct base and 2% of each incorrect base at eachposition within the target were used as one primer, and an oligocorresponding to a non-targeted region of G-luciferase was used as thesecond primer in the PCR reaction. The mismatch library target was thencloned into the dual luciferase reporter in place of the wildtypeG-luciferase using NEB Gibson assembly (New England Biolabs).

Design and Cloning of Mammalian Constructs for RNA Editing

PspCas13b was made catalytically inactive (dPspCas13b) via two histidineto alanine mutations (H133A/H1058A) at the catalytic site of the HEPNdomains. The deaminase domains of human ADAR1 and ADAR2 were synthesizedand PCR amplified for gibson cloning into pcDNA-CMV vector backbones andwere fused to dPspCas13b at the C-terminus via GS or GSGGGGS linkers.For the experiment in which we tested different linkers we cloned thefollowing additional linkers between dPspCas13b and ADAR2dd:GGGGSGGGGSGGGGS (SEQ ID NO: 152), EAAAK, GGSGGSGGSGGSGGSGGS (SEQ ID NO:153), and SGSETPGTSESATPES (XTEN) (SEQ ID NO: 154). Specificity mutantswere generated by gibson cloning the appropriate mutants into thedPspCas13b-GSGGGGS backbone.

The luciferase reporter vector for measuring RNA editing activity wasgenerated by creating a W85X mutation (TGG>TAG) in the luciferasereporter vector used for knockdown experiments. This reporter vectorexpresses functional Gluc as a normalization control, but a defectiveCluc due to the addition of a pretermination site. To test ADAR editingmotif preferences, we cloned every possible motif around the adenosineat codon 85 (XAX) of Cluc.

For testing PFS preference of REPAIR, we cloned a pooled plasmid librarycontaining a 6 basepair degenerate PFS sequence upstream of a targetregion and adenosine editing site. The library was synthesized as anultramer from Integrated DNA Technologies (IDT) and was made doublestranded via annealing a primer and Klenow fragment of DNA polymerase I(New England Biolabs) fill in of the sequence. This dsDNA fragmentcontaining the degenerate sequence was then gibson cloned into thedigested reporter vector and this was then isopropanol precipitated andpurified. The cloned library was then electroporated into Enduracompetent E. coli cells (Lucigen) and plated on 245 mm×245 mm squarebioassay plates (Nunc). After 16 hours, colonies were harvested andmidi-prepped using endotoxin-free MACHEREY-NAGEL midi-prepp kits. Clonedlibraries were verified by next generation sequencing.

For cloning disease-relevant mutations for testing REPAIR activity, 34G>A mutations related to disease pathogenesis as defined in ClinVar wereselected and 200 bp regions surrounding these mutations were golden gatecloned between mScarlett and EGFP under a CMV promoter. Two additionalG>A mutations in AVPR2 and FANCC were selected for Gibson cloning thewhole gene sequence under expression of EF1alpha.

For expression of mammalian guide RNA for REPAIR, the PspCas13b directrepeat sequences were synthesized with golden-gate acceptor sites andcloned under U6 expression via restriction digest cloning. Individualguides were then cloned into this expression backbones by golden gatecloning.

Mammalian Cell Culture

Mammalian cell culture experiments were performed in the HEK293FT line(American Type Culture Collection (ATCC)), which was grown in Dulbecco'sModified Eagle Medium with high glucose, sodium pyruvate, and GlutaMAX(Thermo Fisher Scientific), additionally supplemented with 1×penicillin-streptomycin (Thermo Fisher Scientific) and 10% fetal bovineserum (VWR Seradigm). Cells were maintained at confluency below 80%.

Unless otherwise noted, all transfections were performed withLipofectamine 2000 (Thermo Fisher Scientific) in 96-well plates coatedwith poly-D-lysine (BD Biocoat). Cells were plated at approximately20,000 cells/well sixteen hours prior to transfection to ensure 90%confluency at the time of transfection. For each well on the plate,transfection plasmids were combined with Opti-MEM I Reduced Serum Medium(Thermo Fisher) to a total of 25 μl. Separately, 24.5 ul of Opti-MEM wascombined with 0.5 ul of Lipofectamine 2000. Plasmid and Lipofectaminesolutions were then combined and incubated for 5 minutes, after whichthey were pipetted onto cells.

RNA Knockdown Mammalian Cell Assays

To assess RNA targeting in mammalian cells with reporter constructs, 150ng of Cas13 construct was co-transfected with 300 ng of guide expressionplasmid and 12.5 ng of the knockdown reporter construct. 48 hourspost-transfection, media containing secreted luciferase was removed fromcells, diluted 1:5 in PBS, and measured for activity with BioLuxCypridina and Biolux Gaussia luciferase assay kits (New England Biolabs)on a plate reader (Biotek Synergy Neo2) with an injection protocol. Allreplicates performed are biological replicates.

For targeting of endogenous genes, 150 ng of Cas13 construct wasco-transfected with 300 ng of guide expression plasmid. 48 hourspost-transfection, cells were lysed and RNA was harvested and reversetranscribed using a previously described [CITE PROTOCOLS] modificationof the Cells-to-Ct kit (Thermo Fisher Scientific). cDNA expression wasmeasured via qPCR using TaqMan qPCR probes for the KRAS transcript(Thermo Fisher Scientific), GAPDH control probes (Thermo FisherScientific), and Fast Advanced Master Mix (Thermo Fisher Scientific).qPCR reactions were read out on a LightCycler 480 Instrument II (Roche),with four 5 ul technical replicates in 384-well format.

Evaluation of RNA Specificity Using Pooled Library of Mismatched Targets

The ability of Cas13 to interfere with the mismatched target library wastested using HEK293FT cells seeded in 6 well plates. ˜70% confluentcells were transfected using 2400 ng Cas13 vector, 4800 ng of guide and240 ng of mismatched target library. 48 hours post transfection, cellswere harvested and RNA extracted using the QIAshredder (Qiagen) and theQiagen RNeasy Mini Kit. 1 ug of extracted RNA was reverse transcribedusing the qScript Flex cDNA synthesis kit (Quantabio) following themanufacturer's gene-specific priming protocol and a Gluc specific RTprimer. cDNA was then amplified and sequenced on an Illumina NextSeq.

The sequencing was analyzed by counting reads per sequence and depletionscores were calculated by determining the log 2(−read count ratio)value, where read count ratio is the ratio of read counts in thetargeting guide condition versus the non-targeting guide condition. Thisscore value represents the level of Cas13 activity on the sequence, withhigher values representing stronger depletion and thus higher Cas13cleavage activity. Separate distributions for the single mismatch anddouble mismatch sequences were determined and plotted as heatmaps with adepletion score for each mismatch identity. For double mismatchsequences the average of all possible double mismatches at a givenposition were plotted.

Transcriptome-Wide Profiling of Cas13 in Mammalian Cells by RNASequencing

For measurement of transcriptome-wide specificity, 150 ng of Cas13construct, 300 ng of guide expression plasmid and 15 ng of the knockdownreporter construct were co-transfected; for shRNA conditions, 300 ng ofshRNA targeting plasmid, 15 ng of the knockdown reporter construct, and150 ng of EF1-alpha driven mCherry (to balance reporter load) wereco-transfected. 48 hours after transfection, RNA was purified with theRNeasy Plus Mini kit (Qiagen), mRNA was selected for using NEBNextPoly(A) mRNA Magnetic Isolation Module (New England Biolabs) andprepared for sequencing with the NEBNext Ultra RNA Library Prep Kit forIllumina (New England Biolabs). RNA sequencing libraries were thensequenced on a NextSeq (Illumina).

To analyze transcriptome-wide sequencing data, reads were aligned RefSeqGRCh38 assembly using Bowtie and RSEM version 1.2.31 with defaultparameters [CITE RSEM: accurate transcript quantification from RNA-Seqdata with or without a reference genome]. Transcript expression wasquantified as log 2(TPM+1), genes were filtered for log 2(TPM+1)>2.5 Forselection of differentially expressed genes, only genes withdifferential changes of >2 or <0.75 were considered. Statisticalsignificance of differential expression was evaluated Student's T-teston three targeting replicates versus non-targeting replicates, andfiltered for a false discovery rate of <0.01% by Benj amini-Hochbergprocedure.

ADAR RNA Editing in Mammalian Cells Transfections

To assess REPAIR activity in mammalian cells, we transfected 150 ng ofREPAIR vector, 300 ng of guide expression plasmid, and 40 ng of the RNAediting reporter. After 48 hours, RNA from cells were harvested andreverse transcribed using a method previously described [cite JJ] with agene specific reverse transcription primer. The extracted cDNA was thensubjected to two rounds of PCR to add Illumina adaptors and samplebarcodes using NEBNext High-Fidelity 2×PCR Master Mix. The library wasthen subjected to next generation sequencing on an Illumina NextSeq orMiSeq. RNA editing rates were then evaluated at all adenosine within thesequencing window.

In experiments where the luciferase reporter was targeted for RNAediting, we also harvested the media with secreted luciferase prior toRNA harvest. In this case, because the corrected Cluc might be at lowlevels, we did not dilute the media. We measured luciferase activitywith BioLux Cypridina and Biolux Gaussia luciferase assay kits (NewEngland Biolabs) on a plate reader (Biotek Synergy Neo2) with aninjection protocol. All replicates performed are biological replicates.

PFS Binding Mammalian Screen

To determine the contribution of the PFS to editing efficiency, 625 ngof PFS target library, 4.7 ug of guide, and 2.35 ug of REPAIR wereco-transfected on HEK293FT cells plated in 225 cm2 flasks. Plasmids weremixed with 33 ul of PLUS reagent (Thermo Fisher Scientific), brought to533 ul with Opti-MEM, incubated for 5 minutes, combined with 30 ul ofLipofectamine 2000 and 500 ul of Opti-MEM, incubated for an additional 5minutes, and then pipetted onto cells. 48 hours post-transfection, RNAwas harvested with the RNeasy Plus Mini kit (Qiagen), reversetranscribed with qScript Flex (Quantabio) using a gene specific primer,and amplified with two rounds of PCR using NEBNext High-Fidelity 2×PCRMaster Mix (New England Biolabs) to add Illumina adaptors and samplebarcodes. The library was sequenced on an Illumina NextSeq, and RNAediting rates at the target adenosine were mapped to PFS identity. Toincrease coverage, the PFS was computationally collapsed to 4nucleotides. REPAIR editing rates were calculated for each PFS, averagedover biological replicates with non-targeting rates for thecorresponding PFS subtracted.

Whole-Transcriptome Sequencing to Evaluate ADAR Editing Specificity

For analyzing off-target RNA editing sites across the transcriptome, weharvested total RNA from cells 48 hours post transfection using theRNeasy Plus Miniprep kit (Qiagen). The mRNA fraction is then enrichedusing a NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB) and thisRNA is then prepared for sequencing using NEBNext Ultra RNA Library PrepKit for Illumina (NEB). The libraries were then sequenced on an IlluminaNextSeq and loaded such that there was at least 5 million reads persample.

RNA Editing Analysis for Targeted and Transcriptome Wide Experiments

To analyze the transcriptome-wide RNA editing RNA sequencing data,sequence files were randomly downsampled to 5 million reads. An indexwas generated using the RefSeq GRCh38 assembly with Gluc and Clucsequences added and reads were aligned and quantified using Bowtie/RSEMversion 1.3.0. Alignment BAMs were then sorted and analyzed for RNAediting sites using REDitools [cite] with the following parameters: -t 8-e -d -1-U [AG or TC]-p u -m20 -T6-0 -W -v 1 -n 0.0. Any significantedits found in untransfected or EGFP-transfected conditions wereconsidered to be SNPs or artifacts of the transfection and filtered outfrom the analysis of off-targets. Off-targets were consideredsignificant if the Fisher's exact test yielded a p-value less than 0.5and that at least 2 of 3 biological replicates identified the edit site.

For analyzing the predicted variant effects of each off-target, the listof off-target edit sites was analyzed using the variant annotationintegrator (https://genome.ucsc.edu/cgi-bin/hgVai) as part of the UCSCgenome browser suite of tools using the SIFT and PolyPhen-2 annotations.To declare whether the off-target genes are oncogenic, a database ofoncogenic annotations from the COSMIC catalogue of somatic mutations incancer (cancer.sanger.ac.uk).

For analyzing whether the REPAIR constructs perturbed RNA levels, thetranscript per million (TPM) values output from the RSEM analysis wereused for expression counts and transformed to log-space by taking thelog 2(TPM+1). To find differentially regulated genes, a Student's t-testwas performed on three targeting guide replicates versus threenon-targeting guide replicates. The statistical analysis was onlyperformed on genes with log 2(TPM+1) values greater than 2.5 and geneswere only considered differentially regulated if they had a fold changegreater than 2 or less than 0.8. Genes were reported if they had a falsediscovery rate of less than 0.01.

Results

Comprehensive Characterization of Cas13 Family Members in MammalianCells

We previously developed LwaCas13a for mammalian knockdown applications,but it required an msfGFP stabilization domain for efficient knockdownand, although the specificity was high, knockdown efficiencies were notconsistently below 50%(15). We sought to identify a more robustRNA-targeting CRISPR system by characterizing a genetically diverse setof Cas13 family members to assess their RNA knockdown activity inmammalian cells (FIG. 31A). We cloned 21 Cas13a, 15 Cas13b, and 7 Cas13cmammalian codon-optimized orthologs (Table 6) into an expression vectorwith N- and C-terminal nuclear export signal (NES) sequences and aC-terminal msfGFP to enhance protein stability. To assay interference inmammalian cells, we designed a dual reporter construct expressing theorthogonal Gaussia (Gluc) and Cypridina (Cluc) luciferases underseparate promoters, which allows one luciferase to function as a measureof Cas13 interference activity and the other to serve as an internalcontrol. For each ortholog, we designed PFS-compatible guide RNAs, usingthe Cas13b PFS motifs derived from an ampicillin interference assay(FIG. 55 ; Table 7; Supplementary Note 1) and the 3′ H PFS from previousreports of Cas13a activity(/0).

We transfected HEK293FT cells with Cas13 expression, guide RNA andreporter plasmids and quantified levels of the targeted Gluc 48 hourslater. Testing two guide RNAs for each Cas13 ortholog revealed a rangeof activity levels, including five Cas13b orthologs with similar orincreased interference across both guide RNAs relative to the recentlycharacterized LwaCas13a (FIG. 49B). We selected these five Cas13borthologs, as well as the top two Cas13a orthologs for furtherengineering.

We next tested for Cas13-mediated knockdown of Gluc without msfGFP, inorder to select orthologs that do not require stabilization domains forrobust activity. We hypothesized that, in addition to msfGFP, Cas13activity could be affected by subcellular localization, as previouslyreported for optimization of LwaCas13a(15). Therefore, we tested theinterference activity of the seven selected Cas13 orthologs C-terminallyfused to one of six different localization tags without msfGFP. Usingthe luciferase reporter assay, we found that PspCas13b and PguCas13bC-terminally fused to the HIV Rev gene NES and RanCas13b C-terminallyfused to the MAPK NES had the highest levels of interference activity(FIG. 56A). To further distinguish activity levels of the top orthologs,we compared the three optimized Cas13b constructs to the optimalLwaCas13a-msfGFP fusion and shRNA for their ability to knockdown theKRAS transcript using position-matched guides (FIG. 56B). We observedthe highest levels interference for PspCas13b (average knockdown 62.9%)and thus selected this for further comparison to LwaCas13a.

To more rigorously define the activity level of PspCas13b and LwaCas13awe designed position matched guides tiling along both Gluc and Cluc andassayed their activity using our luciferase reporter assay. We tested 93and 20 position matched guides targeting Gluc and Cluc, respectively,and found that PspCas13b had consistently increased levels of knockdownrelative to LwaCas13a (average of 92.3% for PspCas13b vs. 40.1%knockdown for LwaCas13a) (FIG. 49C,D).

Specificity of Cas13 Mammalian Interference Activity

To characterize the interference specificities of PspCas13b andLwaCas13a we designed a plasmid library of luciferase targets containingsingle mismatches and double mismatches throughout the target sequenceand the three flanking 5′ and 3′ base pairs (FIG. 56C). We transfectedHEK293FT cells with either LwaCas13a or PspCas13b, a fixed guide RNAtargeting the unmodified target sequence, and the mismatched targetlibrary corresponding to the appropriate system. We then performedtargeted RNA sequencing of uncleaved transcripts to quantify depletionof mismatched target sequences. We found that LwaCas13a and PspCas13bhad a central region that was relatively intolerant to singlemismatches, extending from base pairs 12 26 for the PspCas13b target and13-24 for the LwaCas13a target (FIG. 56D). Double mismatches were evenless tolerated than single mutations, with little knockdown activityobserved over a larger window, extending from base pairs 12-29 forPspCas13b and 8-27 for LwaCas13a in their respective targets (FIG. 56E).Additionally, because there are mismatches included in the threenucleotides flanking the 5′ and 3′ ends of the target sequence, we couldassess PFS constraints on Cas13 knockdown activity. Sequencing showedthat almost all PFS combinations allowed robust knockdown, indicatingthat a PFS constraint for interference in mammalian cells likely doesnot exist for either enzyme tested. These results indicate that Cas13aand Cas13b display similar sequence constraints and sensitivitiesagainst mismatches.

We next characterized the interference specificity of PspCas13b andLwaCas13a across the mRNA fraction of the transcriptome. We performedtranscriptome-wide mRNA sequencing to detect significant differentiallyexpressed genes. LwaCas13a and PspCas13b demonstrated robust knockdownof Gluc (FIG. 49E,F) and were highly specific compared to aposition-matched shRNA, which showed hundreds of off-targets (FIG. 49G).

Cas13-ADAR Fusions Enable Targeted RNA Editing

Given that PspCas13b achieved consistent, robust, and specific knockdownof mRNA in mammalian cells, we envisioned that it could be adapted as anRNA binding platform to recruit the deaminase domain of ADARs(ADAR_(DD)) for programmable RNA editing. To engineer a PspCas13blacking nuclease activity (dPspCas13b, referred to as dCas13b fromhere), we mutated conserved catalytic residues in the HEPN domains andobserved loss of luciferase RNA knockdown activity (FIG. 57A). Wehypothesized that a dCas13b-ADAR_(DD) fusion could be recruited by aguide RNA to target adenosines, with the hybridized RNA creating therequired duplex substrate for ADAR activity (FIG. 50A). To enhancetarget adenosine deamination rates we introduced two additionalmodifications to our initial RNA editing design: we introduced amismatched cytidine opposite the target adenosine, which has beenpreviously reported to increase deamination frequency, and fused dCas13bwith the deaminase domains of human ADAR1 or ADAR2 containinghyperactivating mutations to enhance catalytic activity(ADAR1_(DD)(E1008Q)(25) or ADAR2_(DD)(E488Q)(19)).

To test the activity of dCas13b-ADAR_(DD) we generated an RNA-editingreporter on Cluc by introducing a nonsense mutation (W85X (UGG≥UAG)),which could functionally be repaired to the wildtype codon through A≥Iediting (FIG. 50B) and then be detected as restoration of Clucluminescence. We evenly tiled guides with spacers 30, 50, 70 or 84nucleotides in length across the target adenosine to determine theoptimal guide placement and design (FIG. 50C). We found thatdCas13b-ADAR1DD required longer guides to repair the Cluc reporter,while dCas13b-ADAR2_(DD) was functional with all guide lengths tested(FIG. 50C). We also found that the hyperactive E488Q mutation improvedediting efficiency, as luciferase restoration with the wildtypeADAR2_(DD) was reduced (FIG. 57B). From this demonstration of activity,we chose dCas13b-ADAR2_(DD)(E488Q) for further characterization anddesignated this approach as RNA Editing for Programmable A to IReplacement version 1 (REPAIRv1).

To validate that restoration of luciferase activity was due to bona fideediting events, we measured editing of Cluc transcripts subject toREPAIRv1 directly via reverse transcription and targeted next-generationsequencing. We tested 30- and 50-nt spacers around the target site andfound that both guide lengths resulted in the expected A to I edit, with50-nt spacers achieving higher editing percentages (FIG. 50D,E, FIG.57C). We also observed that 50-nt spacers had an increased propensityfor editing at non-targeted adenosines, likely due to increased regionsof duplex RNA (FIG. 50E, FIG. 57C).

We next targeted an endogenous gene, PPIB. We designed 50-nt spacerstiling PPIB and found that we could edit the PPIB transcript with up to28% editing efficiency (FIG. 57D). To test if REPAIR could be furtheroptimized, we modified the linker between dCas13b and ADAR2_(DD)(E488Q)(FIG. 57E, Table 8) and found that linker choice modestly affectedluciferase activity restoration.

Defining the Sequence Parameters for RNA Editing

Given that we could achieve precise RNA editing at a test site, wewanted to characterize the sequence constraints for programming thesystem against any RNA target in the transcriptome. Sequence constraintscould arise from dCas13b targeting limitations, such as the PFS, or fromADAR sequence preferences(26). To investigate PFS constraints onREPAIRv1, we designed a plasmid library carrying a series of fourrandomized nucleotides at the 5′ end of a target site on the Cluctranscript (FIG. 51A). We targeted the center adenosine within either aUAG or AAC motif and found that for both motifs, all PFSs demonstrateddetectable levels of RNA editing, with a majority of the PFSs havinggreater than 50% editing at the target site (FIG. 51B). Next, we soughtto determine if the ADAR2_(DD) in REPAIRv1 had any sequence constraintsimmediately flanking the targeted base, as has been reported previouslyfor ADAR2_(DD)(26). We tested every possible combination of 5′ and 3′flanking nucleotides directly surrounding the target adenosine (FIG.51C), and found that REPAIRv1 was capable of editing all motifs (FIG.51D). Lastly, we analyzed whether the identity of the base opposite thetarget A in the spacer sequence affected editing efficiency and foundthat an A-C mismatch had the highest luciferase restoration with A G,A-U, and A-A having drastically reduced REPAIRv1 activity (FIG. 57F).

Correction of Disease-Relevant Human Mutations Using REPAIRv1

To demonstrate the broad applicability of the REPAIRv1 system for RNAediting in mammalian cells, we designed REPAIRv1 guides against twodisease relevant mutations: 878G>A (AVPR2 W293X) in X-linked Nephrogenicdiabetes insipidus and 1517G>A (FANCC W506X) in Fanconi anemia. Wetransfected expression constructs for cDNA of genes carrying thesemutations into HEK293FT cells and tested whether REPAIRv1 could correctthe mutations. Using guide RNAs containing 50-nt spacers, we were ableto achieve 35% correction of AVPR2 and 23% correction of FANCC (FIG.52A-D). We then tested the ability of REPAIRv1 to correct 34 differentdisease-relevant G>A mutations (Table 9) and found that we were able toachieve significant editing at 33 sites with up to 28% editingefficiency (FIG. 52E). The mutations we chose are only a fraction of thepathogenic G to A mutations (5,739) in the ClinVar database, which alsoincludes an additional 11,943 G to A variants (FIG. 52F and FIG. 58 ).Because there are no sequence constraints, REPAIRv1 is capable ofpotentially editing all these disease relevant mutations, especiallygiven that we observed significant editing regardless of the targetmotif (FIG. 51C and FIG. 52G).

Delivering the REPAIRv1 system to diseased cells is a prerequisite fortherapeutic use, and we therefore sought to design REPAIRv1 constructsthat could be packaged into therapeutically relevant viral vectors, suchas adeno-associated viral (AAV) vectors. AAV vectors have a packaginglimit of 4.7 kb, which cannot accommodate the large size ofdCas13b-ADAR_(DD) (4473 bp) along with promoter and expressionregulatory elements. To reduce the size, we tested a variety ofN-terminal and C-terminal truncations of dCas13 fused toADAR2_(DD)(E488Q) for RNA editing activity. We found that all C-terminaltruncations tested were still functional and able to restore luciferasesignal (FIG. 59 ), and the largest truncation, C-terminal Δ984-1090(total size of the fusion protein 4,152 bp) was small enough to fitwithin the packaging limit of AAV vectors.

Transcriptome-Wide Specificity of REPAIRv1

Although RNA knockdown with PspCas13b was highly specific, in ourluciferase tiling experiments, we observed off-target adenosine editingwithin the guide:target duplex (FIG. 50E). To see if this was awidespread phenomenon, we tiled an endogenous transcript, KRAS, andmeasured the degree of off-target editing near the target adenosine(FIG. 53A). We found that for KRAS, while the on-target editing rate was23%, there were many sites around the target site that also haddetectable A to G edits (FIG. 53B).

Because of the observed off-target editing within the guide:targetduplex, we evaluated all possible transcriptome off-targets byperforming RNA sequencing on all mRNAs. RNA sequencing revealed thatthere was a significant number A to G off-target events, with 1,732off-targets in the targeting condition and 925 off-targets in thenon-targeting condition, with 828 off-targets overlapping (FIG. 53C,D).Of all the editing sites across the transcriptome, the on-target editingsite had the highest editing rate, with 89% A to G conversion.

Given the high specificity of Cas13 targeting, we reasoned that theoff-targets may arise from ADAR. Two RNA-guided ADAR systems have beendescribed previously (FIG. 60A). The first utilizes a fusion ofADAR2_(DD) to the small viral protein lambda N (

N), which binds to the BoxB-

, RNA hairpin(22). A guide RNA with double BoxB

, hairpins guides ADAR2_(DD) to edit sites encoded in the guide RNA(23).The second design utilizes full length ADAR2 (ADAR2) and a guide RNAwith a hairpin that the double strand RNA binding domains (dsRBDs) ofADAR2 recognize(21, 24). We analyzed the editing efficiency of these twosystems compared to REPAIRv1 and found that the BoxB-ADAR2 and ADAR2systems demonstrated 63% and 36% editing rates, respectively, comparedto the 89% editing rate achieved by REPAIRv1 (FIG. 60B-E). Additionally,the BoxB and ADAR2 systems created 2018 and 174 observed off targets,respectively, in the targeting guide conditions, compared to the 1,229off targets in the REPAIRv1 targeting guide condition. Notably, all theconditions with the two ADAR2DD-based systems (REPAIRv1 and BoxB) showeda high percentage of overlap in their off-targets while the ADAR2 systemhad a largely distinct set of off-targets (FIG. 60F). The overlap inoff-targets between the targeting and non-targeting conditions andbetween REPAIRv1 and BoxB conditions suggest ADAR2_(DD) droveoff-targets independent of dCas13 targeting (FIG. 60F).

Improving Specificity of REPAIRv1 Through Rational Protein Engineering

To improve the specificity of REPAIR, we employed structure-guidedprotein engineering of ADAR2_(DD)(E488Q). Because of theguide-independent nature of off-targets, we hypothesized thatdestabilizing ADAR2_(DD)(E488Q)-RNA binding would selectively decreaseoff-target editing, but maintain on-target editing due to increasedlocal concentration from dCas13b tethering of ADAR2_(DD)(E488Q) to thetarget site. We mutagenized ADAR2_(DD)(E488Q) residues previouslydetermined to contact the duplex region of the target RNA (FIG. 54A)(18)on the ADAR2_(DD)(E488Q) background. To assess efficiency andspecificity, we tested 17 single mutants with both targeting andnon-targeting guides, under the assumption that background luciferaserestoration in the non-targeting condition detected would be indicativeof broader off-target activity. We found that mutations at the selectedresidues had significant effects on the luciferase activity fortargeting and non-targeting guides (FIG. 54A,B, FIG. 61A). A majority ofmutants either significantly improved the luciferase activity for thetargeting guide or increased the ratio of targeting to non-targetingguide activity, which we termed the specificity score (FIG. 54A,B). Weselected a subset of these mutants (FIG. 54B) for transcriptome-widespecificity profiling by next generation sequencing. As expected,off-targets measured from transcriptome-wide sequencing correlated withour specificity score (FIG. 61B) for mutants. We found that with theexception of ADAR2_(DD)(E488Q/R455E), all sequenced REPAIRv1 mutantscould effectively edit the reporter transcript (FIG. 54C), with manymutants showing reduction in the number of off-targets (FIGS. 61C, 62 ).We further explored the surrounding motifs of off-targets forspecificity mutants, and found that REPAIRv1 and most of the engineeredmutants exhibited a strong 3′ G preference for their edits, in agreementwith the characterized ADAR2 motif (FIG. 63A)(26). We selected themutant ADAR2_(DD)(E488Q/T375G) for future experiments, as it had thehighest percent editing of the four mutants with the lowest numbers oftranscriptome-wide off targets and termed it REPAIRv2. Compared toREPAIRv1, REPAIRv2 exhibited increased specificity, with a reductionfrom 1732 to 10 transcriptome off-targets (FIG. 54D). In the regionsurrounding the targeted adenosine in Cluc, REPAIRv2 had reducedoff-target editing, visible in sequencing traces (FIG. 54E). In motifsderived from next-generation sequencing, REPAIRv1 presented a strongpreference towards 3′ G, but showed off-targeting edits for all motifs(FIG. 63B); by contrast, REPAIRv2 only edited the strongest off-targetmotifs (FIG. 63C). The distribution of edits on transcripts was heavilyskewed, with highly-edited genes having over 60 edits (FIG. 64A,B),whereas REPAIRv2 only edited one transcript (EEF1A1) multiple times(FIG. 64D-F). REPAIRv1 off-target edits were predicted to result innumerous variants, including 1000 missense mutations (FIG. 64C) with 93oncogenic events (FIG. 64D). In contrast, REPAIRv2 only had 6 missensemutations (FIG. 64E), none of which had oncogenic consequences (FIG.64F). This reduction in predicted off-target effects distinguishesREPAIRv2 from other RNA editing approaches.

We targeted REPAIRv2 to endogenous genes to test if thespecificity-enhancing mutations reduced nearby edits in targettranscripts while maintaining high-efficiency on-target editing. Forguides targeting either KRAS or PPIB, we found that REPAIRv2 had nodetectable off-target edits, unlike REPAIRv1, and could effectively editthe on-target adenosine at 27.1% and 13%, respectively (FIG. 54F,G).This specificity extended to additional target sites, including regionsthat demonstrate high-levels of background in non-targeting conditionsfor REPAIRv1, such as other KRAS or PPIB target sites (FIG. 65 ).Overall, REPAIRv2 eliminated off-targets in duplexed regions around theedited adenosine and showed dramatically enhanced transcriptome-widespecificity.

CONCLUSION

We have shown here that the RNA-guided RNA-targeting type VI-B effectorCas13b is capable of highly efficient and specific RNA knockdown,providing the basis for improved tools for interrogating essential genesand non-coding RNA as well as controlling cellular processes at thetranscriptomic level. Catalytically inactive Cas13b (dCas13b) retainsprogrammable RNA binding capability, which we leveraged here by fusingdCas13b to the adenosine deaminase ADAR2 to achieve precise A to Iedits, a system we term REPAIRv1 (RNA Editing for Programmable A to IReplacement version 1). Further engineering of the system producedREPAIRv2, a method with comparable or increased activity relative tocurrent editing platforms with dramatically improved specificity.

Although Cas13b exhibits high fidelity, our initial results withdCas13b-ADAR2DD fusions revealed thousands of off-targets. To addressthis, we employed a rational mutagenesis strategy to vary the ADAR2_(DD)residues that contact the RNA duplex, identifying a variant,ADAR2_(DD)(E488Q/T375G), capable of precise, efficient, and highlyspecific editing when fused to dCas13b. Editing efficiency with thisvariant was comparable to or better than that achieved with twocurrently available systems, BoxB-ADAR_(DD) or ADAR2 editing. Moreover,the REPAIRv2 system created only 10 observable off-targets in the wholetranscriptome, at least an order of magnitude better than bothalternative editing technologies.

The REPAIR system offers many advantages compared to other nucleic acidediting tools. First, the exact target site can be encoded in the guideby placing a cytidine within the guide extension across from the desiredadenosine to create a favorable A-C mismatch ideal for ADAR editingactivity. Second, Cas13 has no targeting sequence constraints, such as aPFS or PAM, and no motif preference surrounding the target adenosine,allowing any adenosine in the transcriptome to be potentially targetedwith the REPAIR system. We do note, however, that DNA base editors cantarget either the sense or anti-sense strand, while the REPAIR system islimited to transcribed sequences, thereby constraining the total numberof possible editing sites we could target. However, due to the moreflexible nature of targeting with REPAIR, this system can effect moreedits within ClinVar (FIG. 52C) than Cas9-DNA base editors. Third, theREPAIR system directly deaminates target adenosines to inosines and doesnot rely on endogenous repair pathways, such as base-excision ormismatch repair, to generate desired editing outcomes. Thus, REPAIRshould be possible in non-dividing cells that cannot support other formsof editing. Fourth, RNA editing can be transient, allowing the potentialfor temporal control over editing outcomes. This property will likely beuseful for treating diseases caused by temporary changes in cell state,such as local inflammation.

The REPAIR system provides multiple opportunities for additionalengineering. Cas13b possesses pre-crRNA processing activity(13),allowing for multiplex editing of multiple variants, which alone mightnot alter disease risk, but together might have additive effects anddisease-modifying potential. Extension of our rational design approach,such as combining promising mutations, could further increase thespecificity and efficiency of the system, while unbiased screeningapproaches could identify additional residues for improving REPAIRactivity and specificity.

Currently, the base conversions achievable by REPAIR are limited togenerating inosine from adenosine; additional fusions of dCas13 withother catalytic RNA editing domains, such as APOBEC, could enablecytidine to uridine editing. Additionally, mutagenesis of ADAR couldrelax the substrate preference to target cytidine, allowing for theenhanced specificity conferred by the duplexed RNA substrate requirementto be exploited by C-≥U editors. Adenosine to inosine editing on DNAsubstrates may also be possible with catalytically inactiveDNA-targeting CRISPR effectors, such as dCas9 or dCpf1, either throughformation of DNA-RNA heteroduplex targets(27) or mutagenesis of the ADARdomain.

REPAIR could be applied towards a range of therapeutic indications whereA to I (A to G) editing can reverse or slow disease progression (FIG. 66). First, expression of REPAIR for targeting causal, Mendelian G to Amutations in disease-relevant tissues could be used to revertdeleterious mutations and treat disease. For example, stable REPAIRexpression via AAV in brain tissue could be used to correct the GRIN2Amissense mutation c.2191G>A (Asp731Asn) that causes focal epilepsy(28)or the APP missense mutation c.2149G>A (Val717Ile) causing early-onsetAlzheimer's disease(29). Second, REPAIR could be used to treat diseaseby modifying the function of proteins involved in disease-related signaltransduction. For instance, REPAIR editing would allow the re-coding ofsome serine, threonine and tyrosine residues that are the targets ofkinases (FIG. 66 ). Phosphorylation of these residues indisease-relevant proteins affects disease progression for many disordersincluding Alzheimer's disease and multiple neurodegenerativeconditions(30). Third, REPAIR could be used to change the sequence ofexpressed, risk-modifying G to A variants to pre-emptively decrease thechance of entering a disease state for patients. The most intriguingcase are the ‘protective’ risk-modifying alleles, which dramaticallydecrease the chance of entering a disease state, and in some cases,confer additional health benefits. For instance, REPAIR could be used tofunctionally mimic A to G alleles of PCSK9 and IFIH1 that protectagainst cardiovascular disease and psoriatic arthritis(31),respectively. Last, REPAIR can be used to therapeutically modify spliceacceptor and donor sites for exon modulation therapies. REPAIR canchange AU to IU or AA to AI, the functional equivalent of the consensus5′ splice donor or 3′ splice acceptor sites respectively, creating newsplice junctions. Additionally, REPAIR editing can mutate the consensus3′ splice acceptor site from AG-≥IG to promote skipping of the adjacentdownstream exon, a therapeutic strategy that has received significantinterest for the treatment of DMD. Modulation of splice sites could havebroad applications in diseases where anti-sense oligos have had somesuccess, such as for modulation of SMN2 splicing for treatment of spinalmuscular atrophy(32).

We have demonstrated the use of the PspCas13b enzyme as both an RNAknockdown and RNA editing tool. The dCas13b platform for programmableRNA binding has many applications, including live transcript imaging,splicing modification, targeted localization of transcripts, pull downof RNA-binding proteins, and epitranscriptomic modifications. Here, weused dCas13 to create REPAIR, adding to the existing suite of nucleicacid editing technologies. REPAIR provides a new approach for treatinggenetic disease or mimicking protective alleles, and establishes RNAediting as a useful tool for modifying genetic function.

TABLE 6 Cas13 Orthologs used in this study Cas13 Cas13 ID abbreviationHost Organism Protein Accession Cas13a1 LshCas13a Leptotrichia shahiiWP_018451595.1 Cas13a2 LwaCas13a Leptotrichia wadei (Lw2) WP_021746774.1Cas13a3 LseCas13a Listeria seeligeri WP_012985477.1 Cas13a4 LbmCas13aLachnospiraceae bacterium WP_044921188.1 MA2020 Cas13a5 LbnCas13aLachnospiraceae bacterium WP_022785443.1 NK4A179 Cas13a6 CamCas13a[Clostridium] aminophilum DSM WP_031473346.1 10710 Cas13a7 CgaCas13aCarnobacterium gallinarum DSM WP_034560163.1 4847 Cas13a8 Cga2Cas13aCarnobacterium gallinarum DSM WP_034563842.1 4847 Cas13a9 Pprcas13aPaludibacter propionicigenes WP_013443710.1 WB4 Cas13a10 LweCas13aListeria weihenstephanensis FSL WP_036059185.1 R9-0317 Cas13a11LbfCas13a Listeriaceae bacterium FSL M6- WP_036091002.1 0635 Cas13a12Lwa2cas13a Leptotrichia wadei F0279 WP_021746774.1 Cas13a13 RcsCas13aRhodobacter capsulatus SB 1003 WP_013067728.1 Cas13a14 RcrCas13aRhodobacter capsulatus R121 WP_023911507.1 Cas13a15 RcdCas13aRhodobacter capsulatus DE442 WP_023911507.1 Cas13a16 LbuCas13aLeptotrichia buccalis C-1013-b WP_015770004.1 Cas13a17 HheCas13aHerbinix hemicellulosilytica CRZ35554.1 Cas13a18 EreCas13a [Eubacterium]rectale WP_055061018.1 Cas13a19 EbaCas13a Eubacteriaceae bacteriumWP_090127496.1 CHKCI004 Cas13a20 BmaCas13a Blautia sp. Marseille-P2398WP_062808098.1 Cas13a21 LspCas13a Leptotrichia sp. oral taxon 879WP_021744063.1 str. F0557 Cas13b1 BzoCas13b Bergeyella zoohelcumWP_002664492 Cas13b2 PinCas13b Prevotella intermedia WP_036860899Cas13b3 PbuCas13b Prevotella buccae WP_004343973 Cas13b4 AspCas13bAlistipes sp. ZOR0009 WP_047447901 Cas13b5 PsmCas13b Prevotella sp.MA2016 WP_036929175 Cas13b6 RanCas13b Riemerella anatipestiferWP_004919755 Cas13b7 PauCas13b Prevotella aurantiaca WP_025000926Cas13b8 PsaCas13b Prevotella saccharolytica WP_051522484 Cas13b9Pin2Cas13b Prevotella intermedia WP_061868553 Cas13b10 CcaCas13bCapnocytophaga canimorsus WP_013997271 Cas13b11 PguCas13b Porphyromonasgulae WP_039434803 Cas13b12 PspCas13b Prevotella sp. P5-125 WP_044065294Cas13b13 FbrCas13b Flavobacterium branchiophilum WP_014084666 Cas13b14PgiCas13b Porphyromonas gingivalis WP_053444417 Cas13b15 Pin3Cas13bPrevotella intermedia WP_050955369 Cas13c1 FnsCas13c Fusobacteriumnecrophorum WP_005959231.1 subsp. funduliforme ATCC 51357 contig00003Cas13c2 FndCas13c Fusobacterium necrophorum DJ- WP_035906563.1 2contig0065, whole genome shotgun sequence Cas13c3 FnbCas13cFusobacterium necrophorum WP_035935671.1 BFTR-1 contig0068 Cas13c4FnfCas13c Fusobacterium necrophorum EHO19081.1 subsp. funduliforme1_1_36S cont1.14 Cas13c5 FpeCas13c Fusobacterium perfoetens ATCCWP_027128616.1 29250 T364DRAFT_scaffold00009.9_C Cas13c6 FulCas13cFusobacterium ulcerans ATCC WP_040490876.1 49185 cont2.38 Cas13c7AspCas13c Anaerosalibacter sp. ND1 WP_042678931.1 genome assemblyAnaerosalibacter massiliensis ND1

TABLE 7 PFS cutoffs in bacterial screens -Log₂ depletion score used togenerate PFS Cas13b ortholog Key motif Bergeyella zoohelcum 1 2Prevotella intermedia locus 1 2 1 Prevotella buccae 3 3 Alistipes sp.ZOR0009 4 1 Prevotella sp. MA2016 5 2 Riemerella anatipestifer 6 4Prevotella aurantiaca 7 1 Prevotella saccharolytica 8 0 Prevotellaintermedia locus 2 9 0 Capnocytophaga canimorsus 10 3 Porphyromonasgulae 11 4 Prevotella sp. P5-125 12 2.1 Flavobacterium 13 1branchiophilum Porphyromonas gingivalis 14 3 Prevotella intermedia locus2 15 4

TABLE 8 dCas13b-ADAR linker sequences used in thisstudy for RNA editing in mammalian cells. FIG. linker 50C GSGGGGS 50E GS57B GSGGGGS 57C GS 57D GS 57E: GS GS 57E: GSGGGGS GSGGGGS 57E: (GGGS)3GGGGGGGGSGGGGS 57E: Rigid EAAAK 57E: (GGS)6 GGSGGSGGSGGSGGSGGS 57E: XTENSGSETPGTSESATPES 51B GS 57F GS 51C GS 52B GS 52D GS 52E GS51A: Δ984-1090, GS Δ1026-1090, Δ1053-1090 51A: Δ1-125, GSGGGGSΔ1-88, Δ1-72 53B GS 53C GS 53D GS 60A GS 60C GS 60D GS 61D GS 54A GS 62AGS 54B GS 62B GS 62C GS 63A GS 63B GS 54C GS 54D GS 54E GS 54F GS 66A GS66A GS

TABLE 9 Disease information for disease-relevant mutations Full lengthcandidates Gene Disease NM_000054.4(AVPR2):c.878G > A AVPR2 Nephrogenicdiabetes insipidus, (p.Trp293Ter) X-linked NM_000136.2(FANCC):c.1517G >A FANCC Fanconi anemia, (p.Trp506Ter) complementation group C Additionalsimulated candiates Candidate Gene Disease NM_000206.2(IL2RG):c.710G > AIL2RG X-linked severe combined (p.Trp237Ter) immunodeficiencyNM_000132.3(F8):c.3144G > A F8 Hereditary factor VIII (p.Trp1048Ter)deficiency disease NM_000527.4(LDLR):c.1449G > A LDLR Familialhypercholesterolemia (p.Trp483Ter) NM_000071.2(CBS):c.162G > A CBSHomocystinuria due to CBS (p.Trp54Ter) deficiencyNM_000518.4(HBB):c.114G > A HBB beta{circumflex over ( )}0{circumflexover ( )} Thalassemia|beta (p.Trp38Ter) ThalassemiaNM_000035.3(ALDOB):c.888G > A ALDOB Hereditary fructosuria (p.Trp296Ter)NM_004006.2(DMD):c.3747G > A DMD Duchenne muscular dystrophy(p.Trp1249Ter) NM_005359.5(SMAD4):c.906G > A SMAD4 Juvenile polyposissyndrome (p.Trp302Ter) NM_000059.3(BRCA2):c.582G > A BRCA2 Familialcancer of breast|Breast- (p.Trp194Ter) ovarian cancer, familial 2NM_000833.4(GRIN2A):c.3813G > A GRIN2A Epilepsy, focal, with speech(p.Trp1271Ter) disorder and with or without mental retardationNM_002977.3(SCN9A):c.2691G > A SCN9A Indifference to pain, congenital,(p.Trp897Ter) autosomal recessive NM_007375.3(TARDBP):c.943G > A TARDBPAmyotrophic lateral sclerosis (p.Ala315Thr) type 10NM_000492.3(CFTR):c.3846G > A CFTR Cystic fibrosis|Hereditary(p.Trp1282Ter) pancreatitis|not provided|ataluren response- EfficacyNM_130838.1(UBE3A):c.2304G > A UBE3A Angelman syndrome (p.Trp768Ter)NM_000543.4(SMPD1):c.168G > A SMPD1 Niemann-Pick disease, type A(p.Trp56Ter) NM_206933.2(USH2A):c.9390G > A USH2A Usher syndrome, type2A (p.Trp3130Ter) NM_130799.2(MEN1):c.1269G > A MEN1 Hereditarycancer-predisposing (p.Trp423Ter) syndrome NM_177965.3(C8orf37):c.555G >A C8orf37 Retinitis pigmentosa 64 (p.Trp185Ter)NM_000249.3(MLH1):c.1998G > A MLH1 Lynch syndrome (p.Trp666Ter)NM_000548.4(TSC2):c.2108G > A TSC2 Tuberous sclerosis 2|Tuberous(p.Trp703Ter) sclerosis syndrome NM_000267.3(NF1):c.7044G > A NF1Neurofibromatosis, type 1 (p.Trp2348Ter) NM_000179.2(MSH6):c.3020G > AMSH6 Lynch syndrome (p.Trp 1007Ter) NM_000344.3(SMN1):c.305G > A SMN1Spinal muscular atrophy, type (p.Trp102Ter) II|Kugelberg-Welanderdisease NM_024577.3(SH3TC2):c.920G > A SH3TC2 Charcot-Marie-Toothdisease, (p.Trp307Ter) type 4C NM_001369.2(DNAH5):c.8465G > A DNAH5Primary ciliary dyskinesia (p.Trp2822Ter) NM_004992.3(MECP2):c.311G > AMECP2 Rett syndrome (p.Trp 104Ter) NM_032119.3(ADGRV1):c.7406G > AADGRV1 Usher syndrome, type 2C (p.Trp2469Ter)NM_017651.4(AHI1):c.2174G > A AHI1 Joubert syndrome 3 (p.Trp725Ter)NM_004562.2(PRKN):c.1358G > A PRKN Parkinson disease 2 (p.Trp453Ter)NM_000090.3(COL3A1):c.3833G > A COL3A1 Ehlers-Danlos syndrome, type 4(p.Trp1278Ter) NM_007294.3(BRCA1):c.5511G > A BRCA1 Familial cancer ofbreast|Breast- (p.Trp1837Ter) ovarian cancer, familial 1NM_000256.3(MYBPC3):c.3293G > A MYBPC3 Primary familial hypertrophic(p.Trp1098Ter) cardiomyopathy NM_000038.5(APC):c.1262G > A APC Familialadenomatous polyposis (p.Trp421Ter) 1 NM_001204.6(BMPR2):c.893G > ABMPR2 Primary pulmonary (p.W298*) hypertension

TABLE 10 Key plasmids used in this study Plasmid name DescriptionpAB0006 CMV-Cluciferase-polyA EF1a-G-luciferase-polyA pAB0040CMV-Cluciferase(STOP85)-polyA EF1a-G-luciferase-polyA pAB0048pCDNA-ADAR2-N-terminal B12-HIV NES pAB0050 pAB0001-A02 (crRNA backbone)pAB0051 pAB0001-B06 (crRNA backbone) pAB0052 pAB0001-B11 (crRNAbackbone) pAB0053 pAB0001-B12 (crRNA backbone) pAB0014.B6EF1a-BsiWI-Cas13b6-NES-mapk pAB0013.B11 EF1a-BsiWI-Cas13b11-NES-HIVpAB0013.B12 EF1a-BsiWI-Cas13b12-NES-HIV pAB0051 pAB0001-B06 (crRNAbackbone) pAB0052 pAB0001-B11 (crRNA backbone) pAB0053 pAB0001-B12(crRNA backbone) pAB0079 pCDNA-ADAR1hu-EQmutant-N-terminal destinationvector pAB0085 pCDNA-ADAR2 (E488Q)hu-EQmutant-N-terminal destinationvector pAB0095 EF1a-BsiWI-Cas13-B12-NES-HIV, with double H HEPN mutantpAB0114 pCDNA-wtADAR2hu-EQmutant-N-terminal destination vector pAB0120Luciferase ADAR guide optimal (guide 24 from wC0054) pAB0122 pAB0001-B12NT guide for ADAR experiments pAB0151 pCDNA-ADAR2hu-EQmutant-N-terminaldestination vector C-term delta 984-1090 pAB0180 T375G specificitymutant pAB0181 T375G Cas13b C-term delta 984-1090

TABLE 11 Guide/shRNA sequences used in this study forknockdown in mammalian cells Inter- ference Mech- First NameSpacer sequence anism Notes FIG. Bacterial GCCAGCUUUCCGGGCA Cas13bUsed for all PFS UUGGCUUCCAUC orthologs guide (SEQ ID NO: 155) Cas13a-GCCAGCTTTCCGGGCA Cas13a Used for all FIG. Gluc TTGGCTTCCATC Cas13a 49Bguide 1 (SEQ ID NO: 156) orthologs Cas13a- ACCCAGGAATCTCAGG Cas13aUsed for all FIG. Gluc AATGTCGACGAT Cas13a 49B guide 2 (SEQ ID NO: 157)orthologs Cas13a- AGGGTTTTCCCAGTCA Cas13a Used for all FIG. non-CGACGTTGTAAA Cas13a 49B targeting (SEQ ID NO: 158) orthologs guide(LacZ) Cas13b- GGGCATTGGCTTCCAT Cas13b Used for FIG. Gluc CTCTTTGAGCACCTorthologs 49B guide 1.1 (SEQ ID NO: 159) 1-3, 6, 7, 10, 11, 12, 14, 15Cas13b- GUGCAGCCAGCUUUCC Cas13b Used for FIG. Gluc GGGCAUUGGCUUCCortholog 4 49B guide 1.2 (SEQ ID NO: 160) Cas13b- GCAGCCAGCUUUCCGGCas13b Used for FIG. Gluc GCAUUGGCUUCCAU ortholog 5 49B guide 1.3(SEQ ID NO: 161) Cas13b- GGCUUCCAUCUCUUUG Cas13b Used for FIG. GlucAGCACCUCCAGCGG ortholog 8, 49B guide 1.4 (SEQ ID NO: 162) 9 Cas13b-GGAAUGUCGACGAUCG Cas13b Used for FIG. Gluc CCUCGCCUAUGCCG ortholog 1349B guide 1.5 (SEQ ID NO: 163) Cas13b- GAAUGUCGACGAUCGC Cas13b Used forFIG. Gluc CUCGCCUAUGCCGC orthologs 49B guide 2.1 (SEQ ID NO: 164)1-3, 6, 7, 10, 11, 14, 15 Cas13b- GACCUGUGCGAUGAAC Cas13b Used for FIG.Gluc UGCUCCAUGGGCUC ortholog 12 49B guide 2.2 (SEQ ID NO: 165) Cas13b-GUGUGGCAGCGUCCUG Cas13b Used for FIG. Gluc GGAUGAACUUCUUC ortholog 4 49Bguide 2.2 (SEQ ID NO: 166) Cas13b- GUGGCAGCGUCCUGGG Cas13b Used for FIG.Gluc AUGAACUUCUUCAU ortholog 5 49B guide 2.3 (SEQ ID NO: 167) Cas13b-GCUUCUUGCCGGGCAA Cas13b Used for FIG. Gluc CUUCCCGCGGUCAG ortholog 8,49B guide 2.4 (SEQ ID NO: 168) 9 Cas13b- GCAGGGUUUUCCCAGU Cas13bUsed for FIG. Gluc CACGACGUUGUAAAA ortholog 13 49B guide 2.6(SEQ ID NO: 169) Cas13b- GCAGGGUUUUCCCAGU Cas13b Used for all FIG. nonCACGACGUUGUAAAA orthologs 49B targeting (SEQ ID NO: 170) guide Cas13a-ACCCAGGAAUCUCAGG Cas13a FIG. Gluc AAUGUCGACGAU 49E guide-(SEQ ID NO: 171) RNASeq shRNA- CAGCTTTCCGGGCATT shRNA FIG. GlucGGCTT (SEQ ID 49F guide NO: 172) Cas13b- CCGCUGGAGGUGCUCA Cas13b FIG.Gluc AAGAGAUGGAAGCC 49F guide- (SEQ ID NO: 173) RNASeq Cas13a-GCCAGCTTTCCGGGCA Cas13a FIG. Gluc- TTGGCTTCCATC 56A guide-1(SEQ ID NO: 174) Cas13a- ACCCAGGAATCTCAGG Cas13a FIG. Gluc- AATGTCGACGAT56A guide-2 (SEQ ID NO: 175) Cas13b- GGGCATTGGCTTCCAT Cas13b FIG. Gluc-CTCTTTGAGCACCT 56A opt- (SEQ ID NO: 176) guide-1 Cas13b-GAAUGUCGACGAUCGC Cas13b FIG. Gluc- CUCGCCUAUGCCGC 56A opt-(SEQ ID NO: 177) guide-2 Cas13a CAAGGCACTCTTGCCT Cas13a FIG. KRASACGCCACCAGCT 56B guide 1 ((SEQ ID NO: 178) Cas13a TCATATTCGTCCACAACas13a FIG. KRAS AATGATTCTGAA 56B guide 2 (SEQ ID NO: 179) Cas13aATTATTTATGGCAAAT Cas13a FIG. KRAS ACACAAAGAAAG 56B guide 3(SEQ ID NO: 180) Cas13a GAATATCTTCAAATGA Cas13a FIG. KRAS TTTAGTATTATT56B guide 4 (SEQ ID NO: 181) Cas13a ACCATAGGTACATCTT Cas13a FIG. KRASCAGAGTCCTTAA 56B guide 5 (SEQ ID NO: 182) Cas13b GTCAAGGCACTCTTGC Cas13bFIG. KRAS CTACGCCACCAGCT 56B guide 1 (SEQ ID NO: 183) Cas13bGATCATATTCGTCCAC Cas13b FIG. KRAS AAAATGATTCTGAA 56B guide 2(SEQ ID NO: 184) Cas13b GTATTATTTATGGCAA Cas13b FIG. KRAS ATACACAAAGAAAG56B guide 3 (SEQ ID NO: 185) Cas13b GTGAATATCTTCAAAT Cas13b FIG. KRASGATTTAGTATTATT 56B guide 4 (SEQ ID NO: 186) Cas13b GGACCATAGGTACATCCas13b FIG. KRAS TTCAGAGTCCTTAA 56B guide 5 (SEQ ID NO: 187) shRNAaagagtgccttgacga shRNA FIG. KRAS tacagcCTCGAGgctg 56B guide 1tatcgtcaaggcactc tt (SEQ ID NO: 188) shRNA aatcattttgtggacg shRNA FIG.KRAS aatatCTCGAGatatt 56B guide 2 cgtccacaaaatgatt (SEQ ID NO: 189)shRNA aaataatactaaatca shRNA FIG. KRAS tttgaCTCGAGtcaaa 56B guide 3tgatttagtattattt (SEQ ID NO: 190) shRNA aataatactaaatcat shRNA FIG. KRASttgaaCTCGAGttcaa 56B guide 4 atgatttagtattatt (SEQ ID NO: 191) shRNAaaggactctgaagatg shRNA FIG. KRAS tacctCTCGAGaggta 56B guide 5catcttcagagtcctt (SEQ ID NO: 192)

TABLE 12 Guide sequences used for Gluc knockdown First NameSpacer sequence Position Notes FIG. Gluc GAGATCAGGGCAAACA 2Note that the Cas13a 49C tiling GAACTTTGACTCCCspacers are truncated by two guide 1 (SEQ ID NO: 193)nucleotides at the 5′ end Gluc GGATGCAGATCAGGGC 7 Note that the Cas13a49C tiling AAACAGAACTTTGA spacers are truncated by two guide 2(SEQ ID NO: 194) nucleotides at the 5′ end Gluc GCACAGCGATGCAGAT 13Note that the Cas13a 49C tiling CAGGGCAAACAGAAspacers are truncated by two guide 3 (SEQ ID NO: 195)nucleotides at the 5′ end Gluc GCTCGGCCACAGCGAT 19 Note that the Cas13a49C tiling GCAGATCAGGGCAA spacers are truncated by two guide 4(SEQ ID NO: 196) nucleotides at the 5′ end Gluc GGGGCTTGGCCTCGGC 28Note that the Cas13a 49C tiling CACAGCGATGCAGAspacers are truncated by two guide 5 (SEQ ID NO: 197)nucleotides at the 5′ end Gluc GTGGGCTTGGCCTCGG 29 Note that the Cas13a49C tiling CCACAGCGATGCAG spacers are truncated by two guide 6(SEQ ID NO: 198) nucleotides at the 5′ end Gluc GTCTCGGTGGGCTTGG 35Note that the Cas13a 49C tiling CCTCGGCCACAGCGspacers are truncated by two guide 7 (SEQ ID NO: 199)nucleotides at the 5′ end Gluc GTTCGTTGTTCTCGGT 43 Note that the Cas13a49C tiling GGGCTTGGCCTCGG spacers are truncated by two guide 8(SEQ ID NO: 200) nucleotides at the 5′ end Gluc GGAAGTCTTCGTTGTT 49Note that the Cas13a 49C tiling CTCGGTGGGCTTGGspacers are truncated by two guide 9 (SEQ ID NO: 201)nucleotides at the 5′ end Gluc GATGTTGAAGTCTTCG 54 Note that the Cas13a49C tiling TTGTTCTCGGTGGG spacers are truncated by two guide 10(SEQ ID NO: 202) nucleotides at the 5′ end Gluc GCGGCCACGATGTTGA 62Note that the Cas13a 49C tiling AGTCTTCGTTGTTCspacers are truncated by two guide 11 (SEQ ID NO: 203)nucleotides at the 5′ end Gluc GTGGCCACGGCCACGA 68 Note that the Cas13a49C tiling TGTTGAAGTCTTCG spacers are truncated by two guide 12(SEQ ID NO: 204) nucleotides at the 5′ end Gluc GGTTGCTGGCCACGGC 73Note that the Cas13a 49C tiling CACGATGTTGAAGTspacers are truncated by two guide 13 (SEQ ID NO: 205)nucleotides at the 5′ end Gluc GTCGCGAAGTTGCTGG 80 Note that the Cas13a49C tiling CCACGGCCACGATG spacers are truncated by two guide 14(SEQ ID NO: 206) nucleotides at the 5′ end Gluc GCCGTGGTCGCGAAGT 86Note that the Cas13a 49C tiling TGCTGGCCACGGCCspacers are truncated by two guide 15 (SEQ ID NO: 207)nucleotides at the 5′ end Gluc GCGAGATCCGTGGTCG 92 Note that the Cas13a49C tiling CGAAGTTGCTGGCC spacers are truncated by two guide 16(SEQ ID NO: 208) nucleotides at the 5′ end Gluc GCAGCATCGAGATCCG 98Note that the Cas13a 49C tiling TGGTCGCGAAGTTGspacers are truncated by two guide 17 (SEQ ID NO: 209)nucleotides at the 5′ end Gluc GGGTCAGCATCGAGAT 101 Note that the Cas13a49C tiling CCGTGGTCGCGAAG spacers are truncated by two guide 18(SEQ ID NO: 210) nucleotides at the 5′ end Gluc GCTTCCCGCGGTCAGC 109Note that the Cas13a 49C tiling ATCGAGATCCGTGGspacers are truncated by two guide 19 (SEQ ID NO: 211)nucleotides at the 5′ end Gluc GGGGCAACTTCCCGCG 115 Note that the Cas13a49C tiling GTCAGCATCGAGAT spacers are truncated by two guide 20(SEQ ID NO: 212 nucleotides at the 5′ end Gluc GTCTTGCCGGGCAACT 122Note that the Cas13a 49C tiling TCCCGCGGTCAGCAspacers are truncated by two guide 21 (SEQ ID NO: 213)nucleotides at the 5′ end Gluc GGCAGCTTCTTGCCGG 128 Note that the Cas13a49C tiling GCAACTTCCCGCGG spacers are truncated by two guide 22(SEQ ID NO: 214) nucleotides at the 5′ end Gluc GCCAGCGGCAGCTTCT 134Note that the Cas13a 49C tiling TGCCGGGCAACTTCspacers are truncated by two guide 23 (SEQ ID NO: 215)nucleotides at the 5′ end Gluc GCACCTCCAGCGGCAG 139 Note that the Cas13a49C tiling CTTCTTGCCGGGCA spacers are truncated by two guide 24(SEQ ID NO: 216) nucleotides at the 5′ end Gluc GCTTTGAGCACCTCCA 146Note that the Cas13a 49C tiling GCGGCAGCTTCTTGspacers are truncated by two guide 25 (SEQ ID NO: 217)nucleotides at the 5′ end Gluc GCATCTCTTTGAGCAC 151 Note that the Cas13a49C tiling CTCCAGCGGCAGCT spacers are truncated by two guide 26(SEQ ID NO: 218) nucleotides at the 5′ end Gluc GTCCATCTCTTTGAGC 153Note that the Cas13a 49C tiling ACCTCCAGCGGCAGspacers are truncated by two guide 27 (SEQ ID NO: 219)nucleotides at the 5′ end Gluc GGGCATTGGCTTCCAT 163 Note that the Cas13a49C tiling CTCTTTGAGCACCT spacers are truncated by two guide 28(SEQ ID NO: 220) nucleotides at the 5′ end Gluc GTCCGGGCATTGGCTT 167Note that the Cas13a 49C tiling CCATCTCTTTGAGCspacers are truncated by two guide 29 (SEQ ID NO: 221)nucleotides at the 5′ end Gluc GGCCAGCTTTCCGGGC 175 Note that the Cas13a49C tiling ATTGGCTTCCATCT spacers are truncated by two guide 30(SEQ ID NO: 222) nucleotides at the 5′ end Gluc GGGTGCAGCCAGCTTT 181Note that the Cas13a 49C tiling CCGGGCATTGGCTTspacers are truncated by two guide 31 (SEQ ID NO: 223)nucleotides at the 5′ end Gluc GAGCCCCTGGTGCAGC 188 Note that the Cas13a49C tiling CAGCTTTCCGGGCA spacers are truncated by two guide 32(SEQ ID NO: 224) nucleotides at the 5′ end Gluc GATCAGACAGCCCCTG 195Note that the Cas13a 49C tiling GTGCAGCCAGCTTTspacers are truncated by two guide 33 (SEQ ID NO: 225)nucleotides at the 5′ end Gluc GGCAGATCAGACAGCC 199 Note that the Cas13a49C tiling CCTGGTGCAGCCAG spacers are truncated by two guide 34(SEQ ID NO: 226) nucleotides at the 5′ end Gluc GACAGGCAGATCAGAC 203Note that the Cas13a 49C tiling AGCCCCTGGTGCAGspacers are truncated by two guide 35 (SEQ ID NO: 227)nucleotides at the 5′ end Gluc GTGATGTGGGACAGGC 212 Note that the Cas13a49C tiling AGATCAGACAGCCC spacers are truncated by two guide 36(SEQ ID NO: 228) nucleotides at the 5′ end Gluc GACTTGATGTGGGACA 215Note that the Cas13a 49C tiling GGCAGATCAGACAGspacers are truncated by two guide 37 (SEQ ID NO: 229)nucleotides at the 5′ end Gluc GGGGCGTGCACTTGAT 223 Note that the Cas13a49C tiling GTGGGACAGGCAGA spacers are truncated by two guide 38(SEQ ID NO: 230) nucleotides at the 5′ end Gluc GCTTCATCTTGGGCGT 232Note that the Cas13a 49C tiling GCACTTGATGTGGGspacers are truncated by two guide 39 (SEQ ID NO: 231)nucleotides at the 5′ end Gluc GTGAACTTCTTCATCT 239 Note that the Cas13a49C tiling TGGGCGTGCACTTG spacers are truncated by two guide 40(SEQ ID NO: 232) nucleotides at the 5′ end Gluc GGGATGAACTTCTTCA 242Note that the Cas13a 49C tiling TCTTGGGCGTGCACspacers are truncated by two guide 41 (SEQ ID NO: 233)nucleotides at the 5′ end Gluc GTGGGATGAACTTCTT 244 Note that the Cas13a49C tiling CATCTTGGGCGTGC spacers are truncated by two guide 42(SEQ ID NO: 234) nucleotides at the 5′ end Gluc GGGCAGCGTCCTGGGA 254Note that the Cas13a 49C tiling TGAACTTCTTCATCspacers are truncated by two guide 43 (SEQ ID NO: 235)nucleotides at the 5′ end Gluc GGGTGTGGCAGCGTCC 259 Note that the Cas13a49C tiling TGGGATGAACTTCT spacers are truncated by two guide 44(SEQ ID NO: 236) nucleotides at the 5′ end Gluc GTTCGTAGGTGTGGCA 265Note that the Cas13a 49C tiling GCGTCCTGGGATGAspacers are truncated by two guide 45 (SEQ ID NO: 237)nucleotides at the 5′ end Gluc GCGCCTTCGTAGGTGT 269 Note that the Cas13a49C tiling GGCAGCGTCCTGGG spacers are truncated by two guide 46(SEQ ID NO: 238) nucleotides at the 5′ end Gluc GTCTTTGTCGCCTTCG 276Note that the Cas13a 49C tiling TAGGTGTGGCAGCGspacers are truncated by two guide 47 (SEQ ID NO: 239)nucleotides at the 5′ end Gluc GCTTTGTCGCCTTCGT 275 Note that the Cas13a49C tiling AGGTGTGGCAGCGT spacers are truncated by two guide 48(SEQ ID NO: 240) nucleotides at the 5′ end Gluc GTGCCGCCCTGTGCGG 293Note that the Cas13a 49C tiling ACTCTTTGTCGCCTspacers are truncated by two guide 49 (SEQ ID NO: 241)nucleotides at the 5′ end Gluc GTATGCCGCCCTGTGC 295 Note that the Cas13a49C tiling GGACTCTTTGTCGC spacers are truncated by two guide 50(SEQ ID NO: 242) nucleotides at the 5′ end Gluc GCCTCGCCTATGCCGC 302Note that the Cas13a 49C tiling CCTGTGCGGACTCTspacers are truncated by two guide 51 (SEQ ID NO: 243)nucleotides at the 5′ end Gluc GGATCGCCTCGCCTAT 307 Note that the Cas13a49C tiling GCCGCCCTGTGCGG spacers are truncated by two guide 52(SEQ ID NO: 244) nucleotides at the 5′ end Gluc GATGTCGACGATCGCC 315Note that the Cas13a 49C tiling TCGCCTATGCCGCCspacers are truncated by two guide 53 (SEQ ID NO: 245)nucleotides at the 5′ end Gluc GCAGGAATGTCGACGA 320 Note that the Cas13a49C tiling TCGCCTCGCCTATG spacers are truncated by two guide 54(SEQ ID NO: 246) nucleotides at the 5′ end Gluc GAATCTCAGGAATGTC 325Note that the Cas13a 49C tiling GACGATCGCCTCGCspacers are truncated by two guide 55 (SEQ ID NO: 247)nucleotides at the 5′ end Gluc GCCCAGGAATCTCAGG 331 Note that the Cas13a49C tiling AATGTCGACGATCG spacers are truncated by two guide 56(SEQ ID NO: 248) nucleotides at the 5′ end Gluc GCCTTGAACCCAGGAA 338Note that the Cas13a 49C tiling TCTCAGGAATGTCGspacers are truncated by two guide 57 (SEQ ID NO: 249)nucleotides at the 5′ end Gluc GCCAAGTCCTTGAACC 344 Note that the Cas13a49C tiling CAGGAATCTCAGGA spacers are truncated by two guide 58(SEQ ID NO: 250) nucleotides at the 5′ end Gluc GTGGGCTCCAAGTCCT 350Note that the Cas13a 49C tiling TGAACCCAGGAATCspacers are truncated by two guide 59 (SEQ ID NO: 251)nucleotides at the 5′ end Gluc GCCATGGGCTCCAAGT 353 Note that the Cas13a49C tiling CCTTGAACCCAGGA spacers are truncated by two guide 60(SEQ ID NO: 252) nucleotides at the 5′ end Gluc GGAACTGCTCCATGGG 361Note that the Cas13a 49C tiling CTCCAAGTCCTTGAspacers are truncated by two guide 61 (SEQ ID NO: 253)nucleotides at the 5′ end Gluc GTGCGATGAACTGCTC 367 Note that the Cas13a49C tiling CATGGGCTCCAAGT spacers are truncated by two guide 62(SEQ ID NO: 254) nucleotides at the 5′ end Gluc GGACCTGTGCGATGAA 373Note that the Cas13a 49C tiling CTGCTCCATGGGCTspacers are truncated by two guide 63 (SEQ ID NO: 255)nucleotides at the 5′ end Gluc GACAGATCGACCTGTG 380 Note that the Cas13a49C tiling CGATGAACTGCTCC spacers are truncated by two guide 64(SEQ ID NO: 256) nucleotides at the 5′ end Gluc GACACACAGATCGACC 384Note that the Cas13a 49C tiling TGTGCGATGAACTGspacers are truncated by two guide 65 (SEQ ID NO: 257)nucleotides at the 5′ end Gluc GTGCAGTCCACACACA 392 Note that the Cas13a49C tiling GATCGACCTGTGCG spacers are truncated by two guide 66(SEQ ID NO: 258) nucleotides at the 5′ end Gluc GCCAGTTGTGCAGTCC 399Note that the Cas13a 49C tiling ACACACAGATCGACspacers are truncated by two guide 67 (SEQ ID NO: 259)nucleotides at the 5′ end Gluc GGGCAGCCAGTTGTGC 404 Note that the Cas13a49C tiling AGTCCACACACAGA spacers are truncated by two guide 68(SEQ ID NO: 260) nucleotides at the 5′ end Gluc GTTTGAGGCAGCCAGT 409Note that the Cas13a 49C tiling TGTGCAGTCCACACspacers are truncated by two guide 69 (SEQ ID NO: 261)nucleotides at the 5′ end Gluc GAAGCCCTTTGAGGCA 415 Note that the Cas13a49C tiling GCCAGTTGTGCAGT spacers are truncated by two guide 70(SEQ ID NO: 262) nucleotides at the 5′ end Gluc GCACGTTGGCAAGCCC 424Note that the Cas13a 49C tiling TTTGAGGCAGCCAGspacers are truncated by two guide 71 (SEQ ID NO: 263)nucleotides at the 5′ end Gluc GACTGCACGTTGGCAA 428 Note that the Cas13a49C tiling GCCCTTTGAGGCAG spacers are truncated by two guide 72(SEQ ID NO: 264) nucleotides at the 5′ end Gluc GGGTCAGAACACTGCA 437Note that the Cas13a 49C tiling CGTTGGCAAGCCCTspacers are truncated by two guide 73 (SEQ ID NO: 265)nucleotides at the 5′ end Gluc GCAGGTCAGAACACTG 439 Note that the Cas13a49C tiling CACGTTGGCAAGCC spacers are truncated by two guide 74(SEQ ID NO: 266) nucleotides at the 5′ end Gluc GAGCAGGTCAGAACAC 441Note that the Cas13a 49C tiling TGCACGTTGGCAAGspacers are truncated by two guide 75 (SEQ ID NO: 267)nucleotides at the 5′ end Gluc GGCCACTTCTTGAGCA 452 Note that the Cas13a49C tiling GGTCAGAACACTGC spacers are truncated by two guide 76(SEQ ID NO: 268) nucleotides at the 5′ end Gluc GCGGCAGCCACTTCTT 457Note that the Cas13a 49C tiling GAGCAGGTCAGAACspacers are truncated by two guide 77 (SEQ ID NO: 269)nucleotides at the 5′ end Gluc GTGCGGCAGCCACTTC 459 Note that the Cas13a49C tiling TTGAGCAGGTCAGA spacers are truncated by two guide 78(SEQ ID NO: 270) nucleotides at the 5′ end Gluc GAGCGTTGCGGCAGCC 464Note that the Cas13a 49C tiling ACTTCTTGAGCAGGspacers are truncated by two guide 79 (SEQ ID NO: 271)nucleotides at the 5′ end Gluc GAAAGGTCGCACAGCG 475 Note that the Cas13a49C tiling TTGCGGCAGCCACT spacers are truncated by two guide 80(SEQ ID NO: 272) nucleotides at the 5′ end Gluc GCTGGCAAAGGTCGCA 480Note that the Cas13a 49C tiling CAGCGTTGCGGCAGspacers are truncated by two guide 81 (SEQ ID NO: 273)nucleotides at the 5′ end Gluc GGGCAAAGGTCGCACA 478 Note that the Cas13a49C tiling GCGTTGCGGCAGCC spacers are truncated by two guide 82(SEQ ID NO: 274) nucleotides at the 5′ end Gluc GTGGATCTTGCTGGCA 489Note that the Cas13a 49C tiling AAGGTCGCACAGCGspacers are truncated by two guide 83 (SEQ ID NO: 275)nucleotides at the 5′ end Gluc GCACCTGGCCCTGGAT 499 Note that the Cas13a49C tiling CTTGCTGGCAAAGG spacers are truncated by two guide 84(SEQ ID NO: 276) nucleotides at the 5′ end Gluc GTGGCCCTGGATCTTG 495Note that the Cas13a 49C tiling CTGGCAAAGGTCGCspacers are truncated by two guide 85 (SEQ ID NO: 277)nucleotides at the 5′ end Gluc GTGATCTTGTCCACCT 509 Note that the Cas13a49C tiling GGCCCTGGATCTTG spacers are truncated by two guide 86(SEQ ID NO: 278) nucleotides at the 5′ end Gluc GCCCCTTGATCTTGTC 514Note that the Cas13a 49C tiling CACCTGGCCCTGGAspacers are truncated by two guide 87 (SEQ ID NO: 279)nucleotides at the 5′ end Gluc GCCCTTGATCTTGTCC 513 Note that the Cas13a49C tiling ACCTGGCCCTGGAT spacers are truncated by two guide 88(SEQ ID NO: 280) nucleotides at the 5′ end Gluc GCCTTGATCTTGTCCA 512Note that the Cas13a 49C tiling CCTGGCCCTGGATCspacers are truncated by two guide 89 (SEQ ID NO: 281)nucleotides at the 5′ end Gluc GGCAAAGGTCGCACAG 477 Note that the Cas13a49C tiling CGTTGCGGCAGCCA spacers are truncated by two guide 90(SEQ ID NO: 282) nucleotides at the 5′ end Gluc GCAAAGGTCGCACAGC 476Note that the Cas13a 49C tiling GTTGCGGCAGCCACspacers are truncated by two guide 91 (SEQ ID NO: 283)nucleotides at the 5′ end Gluc GAAGGTCGCACAGCGT 474 Note that the Cas13a49C tiling TGCGGCAGCCACTT spacers are truncated by two guide 92(SEQ ID NO: 284) nucleotides at the 5′ end Gluc GAGGTCGCACAGCGTT 473Note that the Cas13a 49C tiling GCGGCAGCCACTTCspacers are truncated by two guide 93 (SEQ ID NO: 285)nucleotides at the 5′ end Non- GGTAATGCCTGGCTTG N/A Note that the Cas13a49C targeting TCGACGCATAGTCTG spacers are truncated by two guide 1(SEQ ID NO: 286) nucleotides at the 5′ end Non- GGGAACCTTGGCCGTT N/ANote that the Cas13a 49C targeting ATAAAGTCTGACCAGspacers are truncated by two guide 2 (SEQ ID NO: 287)nucleotides at the 5′ end Non- GGAGGGTGAGAATTTA N/A Note that the Cas13a49C targeting GAACCAAGATTGTTG spacers are truncated by two guide 3(SEQ ID NO: 288) nucleotides at the 5′ end

TABLE 13 Guide sequences used for Cluc knockdown First NameSpacer sequence Position Notes FIG. Cluc GAGTCCTGGCAATGA   32Note that the Cas13a 49D tiling ACAGTGGCGCAGTAGspacers are truncated by two guide 1 (SEQ ID NO: 289)nucleotides at the 5′ end Cluc GGGTGCCACAGCTGC  118 Note that the Cas13a49D tiling TATCAATACATTCTC spacers are truncated by two guide 2(SEQ ID NO: 290) nucleotides at the 5′ end Cluc GTTACATACTGACAC  197Note that the Cas13a 49D tiling ATTCGGCAACATGTTspacers are truncated by two guide 3 (SEQ ID NO: 291)nucleotides at the 5′ end Cluc GTATGTACCAGGTTC  276 Note that the Cas13a49D tiling CTGGAACTGGAATCT spacers are truncated by two guide 4(SEQ ID NO: 292) nucleotides at the 5′ end Cluc GCCTTGGTTCCATCC  350Note that the Cas13a 49D tiling AGGTTCTCCAGGGTGspacers are truncated by two guide 5 (SEQ ID NO: 293)nucleotides at the 5′ end Cluc GCAGTGATGGGATTC  431 Note that the Cas13a49D tiling TCAGTAGCTTGAGCG spacers are truncated by two guide 6(SEQ ID NO: 294) nucleotides at the 5′ end Cluc GAGCCTGGCATCTCA  512Note that the Cas13a 49D tiling ACAACAGCGATGGTGspacers are truncated by two guide 7 (SEQ ID NO: 295)nucleotides at the 5′ end Cluc GTGTCTGGGGCGATT  593 Note that the Cas13a49D tiling CTTACAGATCTTCCT spacers are truncated by two guide 8(SEQ ID NO: 296) nucleotides at the 5′ end Cluc GCTGGATCTGAAGTG  671Note that the Cas13a 49D tiling AAGTCTGTATCTTCCspacers are truncated by two guide 9 (SEQ ID NO: 297)nucleotides at the 5′ end Cluc GGCAACGTCATCAGG  747 Note that the Cas13a49D tiling ATTTCCATAGAGTGG spacers are truncated by two guide 10(SEQ ID NO: 298) nucleotides at the 5′ end Cluc GAGGCGCAGGAGATG  830Note that the Cas13a 49D tiling GTGTAGTAGTAGAAGspacers are truncated by two guide 11 (SEQ ID NO: 299)nucleotides at the 5′ end Cluc GAGGGACCCTGGAAT  986 Note that the Cas13a49D tiling TGGTATCTTGCTTTG spacers are truncated by two guide 13(SEQ ID NO: 300) nucleotides at the 5′ end Cluc GGTAAGAGTCAACAT 1066Note that the Cas13a 49D tiling TCCTGTGTGAAACCTspacers are truncated by two guide 14 (SEQ ID NO: 301)nucleotides at the 5′ end Cluc GACCAGAATCTGTTT 1143 Note that the Cas13a49D tiling TCCATCAACAATGAG spacers are truncated by two guide 15(SEQ ID NO: 302) nucleotides at the 5′ end Cluc GATGGCTGTAGTCAG 1227Note that the Cas13a 49D tiling TATGTCACCATCTTGspacers are truncated by two guide 16 (SEQ ID NO: 303)nucleotides at the 5′ end Cluc GTACCATCGAATGGA 1304 Note that the Cas13a49D tiling TCTCTAATATGTACG spacers are truncated by two guide 17(SEQ ID NO: 304) nucleotides at the 5′ end Cluc GAGATCACAGGCTCC 1380Note that the Cas13a 49D tiling TTCAGCATCAAAAGAspacers are truncated by two guide 18 (SEQ ID NO: 305)nucleotides at the 5′ end Cluc GCTTTGACCGGCGAA 1461 Note that the Cas13a49D tiling GAGACTATTGCAGAG spacers are truncated by two guide 19(SEQ ID NO: 306) nucleotides at the 5′ end Cluc GCCCCTCAGGCAATA 1539Note that the Cas13a 49D tiling CTCGTACATGCATCGspacers are truncated by two guide 20 (SEQ ID NO: 307)nucleotides at the 5′ end Cluc GCTGGTACTTCTAGG 1619 Note that the Cas13a49D tiling GTGTCTCCATGCTTT spacers are truncated by two guide 21(SEQ ID NO: 308) nucleotides at the 5′ end Non- GGTAATGCCTGGCTTG N/ANote that the Cas13a 49D targeting TCGACGCATAGTCTGspacers are truncated by two guide 1 (SEQ ID NO: 309)nucleotides at the 5′ end Non- GGGAACCTTGGCCGTT N/A Note that the Cas13a49D targeting ATAAAGTCTGACCAG spacers are truncated by two guide 2(SEQ ID NO: 310) nucleotides at the 5′ end Non- GGAGGGTGAGAATTTA N/ANote that the Cas13a 49D targeting GAACCAAGATTGTTGspacers are truncated by two guide 3 (SEQ ID NO: 311)nucleotides at the 5′ end

TABLE 14Guide sequences used in this study for RNA editing in mammalian cells.Mismatched base flips are capitalized First Name Spacer sequence NotesFIG. Tiling 30 nt 30 gCatcctgcggcctctactctgcattcaat Has a 5′ G for U650C mismatch (SEQ ID NO: 312) expression distance Tiling 30 nt 28gacCatcctgcggcctctactctgcattca Has a 5′ G for U6 50C mismatcha (SEQ ID NO: 313) expression distance Tiling 30 nt 26gaaacCatcctgcggcctctactctgcatt Has a 5′ G for U6 50C mismatchc (SEQ ID NO: 314) expression distance Tiling 30 nt 24gctaaacCatcctgcggcctctactctgca Has a 5′ G for U6 50C mismatcht (SEQ ID NO: 315) expression distance Tiling 30 nt 22gttctaaacCatcctgcggcctctactctg Has a 5′ G for U6 50C mismatchc (SEQ ID NO: 316) expression distance Tiling 30 nt 20gtgttctaaacCatcctgcggcctctactct Has a 5′ G for U6 50C mismatch(SEQ ID NO: 317) expression distance Tiling 30 nt 18gaatgttctaaacCatcctgcggcctctac Has a 5′ G for U6 50C mismatcht (SEQ ID NO: 318) expression distance Tiling 30 nt 16gagaatgttctaaacCatcctgcggcctct Has a 5′ G for U6 50C mismatcha (SEQ ID NO: 319) expression distance Tiling 30 nt 14gatagaatgttctaaacCatcctgcggcct Has a 5′ G for U6 50C mismatchc (SEQ ID NO: 320) expression distance Tiling 30 nt 12gccatagaatgttctaaacCatcctgcgg Has a 5′ G for U6 50C mismatchcc (SEQ ID NO: 321) expression distance Tiling 30 nt 10gttccatagaatgttctaaacCatcctgcg Has a 5′ G for U6 50C mismatchg (SEQ ID NO: 322) expression distance Tiling 30 nt 8gctttccatagaatgttctaaacCatcctgc Has a 5′ G for U6 50C mismatch(SEQ ID NO: 323) expression distance Tiling 30 nt 6gctctttccatagaatgttctaaacCatcct Has a 5′ G for U6 50C mismatch(SEQ ID NO: 324) expression distance Tiling 30 nt 4gatctctttccatagaatgttctaaacCatc Has a 5′ G for U6 50C mismatch(SEQ ID NO: 325) expression distance Tiling 30 nt 2ggaatctctttccatagaatgttctaaacCa Has a 5′ G for U6 50C mismatch(SEQ ID NO: 326) expression distance Tiling 50 nt 50gCatcctgcggcctctactctgcattcaatt Has a 5′ G for U6 50C mismatchacatactgacacattcggca (SEQ ID expression distance NO: 327)Tiling 50 nt 48 gacCatcctgcggcctctactctgcattca Has a 5′ G for U6 50Cmismatch attacatactgacacattcgg (SEQ ID expression distance NO: 328)Tiling 50 nt 46 gaaacCatcctgcggcctctactctgcatt Has a 5′ G for U6 50Cmismatch caattacatactgacacattc (SEQ ID expression distance NO: 329)Tiling 50 nt 44 gctaaacCatcctgcggcctctactctgca Has a 5′ G for U6 50Cmismatch ttcaattacatactgacacat (SEQ ID expression distance NO: 330)Tiling 50 nt 42 gttctaaacCatcctgcggcctctactctg Has a 5′ G for U6 50Cmismatch cattcaattacatactgacac (SEQ ID expression distance NO: 331)Tiling 50 nt 40 gtgttctaaacCatcctgcggcctctactct Has a 5′ G for U6 50Cmismatch gcattcaattacatactgac (SEQ ID expression distance NO: 332)Tiling 50 nt 38 gaatgttctaaacCatcctgcggcctctac Has a 5′ G for U6 50Cmismatch tctgcattcaattacatactg (SEQ ID expression distance NO: 333)Tiling 50 nt 36 gagaatgttctaaacCatcctgcggcctct Has a 5′ G for U6 50Cmismatch actctgcattcaattacatac (SEQ ID expression distance NO: 334)Tiling 50 nt 34 gatagaatgttctaaacCatcctgcggcct Has a 5′ G for U6 50Cmismatch ctactctgcattcaattacat (SEQ ID expression distance NO: 335)Tiling 50 nt 32 gccatagaatgttctaaacCatcctgcgg Has a 5′ G for U6 50Cmismatch cctctactctgcattcaattac (SEQ ID expression distance NO: 336)Tiling 50 nt 30 gttccatagaatgttctaaacCatcctgcg Has a 5′ G for U6 50Cmismatch gcctctactctgcattcaatt (SEQ ID expression distance NO: 337)Tiling 50 nt 28 gctttccatagaatgttctaaacCatcctgc Has a 5′ G for U6 50Cmismatch ggcctctactctgcattcaa (SEQ ID expression distance NO: 338)Tiling 50 nt 26 gctctttccatagaatgttctaaacCatcct Has a 5′ G for U6 50Cmismatch gcggcctctactctgcattc (SEQ ID expression distance NO: 339)Tiling 50 nt 24 gatctctttccatagaatgttctaaacCatc Has a 5′ G for U6 50Cmismatch ctgcggcctctactctgcat (SEQ ID expression distance NO: 340)Tiling 50 nt 22 ggaatctctttccatagaatgttctaaacCa Has a 5′ G for U6 50Cmismatch tcctgcggcctctactctgc (SEQ ID expression distance NO: 341)Tiling 50 nt 20 gtggaatctctttccatagaatgttctaaac Has a 5′ G for U6 50Cmismatch Catcctgcggcctctactct (SEQ ID expression distance NO: 342)Tiling 50 nt 18 gactggaatctctttccatagaatgttctaaa Has a 5′ G for U6 50Cmismatch cCatcctgcggcctctact (SEQ ID expression distance NO: 343)Tiling 50 nt 16 ggaactggaatctctttccatagaatgttct Has a 5′ G for U6 50Cmismatch aaacCatcctgcggcctcta (SEQ ID expression distance NO: 344)Tiling 50 nt 14 gtggaactggaatctctttccatagaatgtt Has a 5′ G for U6 50Cmismatch ctaaacCatcctgcggcctc (SEQ ID expression distance NO: 345)Tiling 50 nt 12 gcctggaactggaatctctttccatagaatg Has a 5′ G for U6 50Cmismatch ttctaaacCatcctgcggcc (SEQ ID expression distance NO: 346)Tiling 50 nt 10 gttcctggaactggaatctctttccatagaa Has a 5′ G for U6 50Cmismatch tgttctaaacCatcctgcgg (SEQ ID expression distance NO: 347)Tiling 50 nt 8 gggttcctggaactggaatctctttccatag Has a 5′ G for U6 50Cmismatch aatgttctaaacCatcctgc (SEQ ID expression distance NO: 348)Tiling 50 nt 6 gcaggttcctggaactggaatctctttccat Has a 5′ G for U6 50Cmismatch agaatgttctaaacCatcct (SEQ ID expression distance NO: 349)Tiling 50 nt 4 gaccaggttcctggaactggaatctctttcc Has a 5′ G for U6 50Cmismatch atagaatgttctaaacCatc (SEQ ID expression distance NO: 350)Tiling 50 nt 2 ggtaccaggttcctggaactggaatctcttt Has a 5′ G for U6 50Cmismatch ccatagaatgttctaaacCa (SEQ ID expression distance NO: 351)Tiling 70 nt 70 gCatcctgcggcctctactctgcattcaatt Has a 5′ G for U6 50Cmismatch acatactgacacattcggcaacatgtttttc expression distancectggtttat (SEQ ID NO: 352) Tiling 70 nt 68gacCatcctgcggcctctactctgcattca Has a 5′ G for U6 50C mismatchattacatactgacacattcggcaacatgtttt expression distancetcctggttt (SEQ ID NO: 353) Tiling 70 nt 66gaaacCatcctgcggcctctactctgcatt Has a 5′ G for U6 50C mismatchcaattacatactgacacattcggcaacatgt expression distancettttcctggt (SEQ ID NO: 354) Tiling 70 nt 64gctaaacCatcctgcggcctctactctgca Has a 5′ G for U6 50C mismatchttcaattacatactgacacattcggcaacat expression distancegtttttcctg (SEQ ID NO: 355) Tiling 70 nt 62gttctaaacCatcctgcggcctctactctg Has a 5′ G for U6 50C mismatchcattcaattacatactgacacattcggcaac expression distanceatgtttttcc (SEQ ID NO: 356) Tiling 70 nt 60gtgttctaaacCatcctgcggcctctactct Has a 5′ G for U6 50C mismatchgcattcaattacatactgacacattcggcaa expression distancecatgttttt (SEQ ID NO: 357) Tiling 70 nt 58gaatgttctaaacCatcctgcggcctctac Has a 5′ G for U6 50C mismatchtctgcattcaattacatactgacacattcgg expression distancecaacatgttt (SEQ ID NO: 358) Tiling 70 nt 56gagaatgttctaaacCatcctgcggcctct Has a 5′ G for U6 50C mismatchactctgcattcaattacatactgacacattc expression distanceggcaacatgt (SEQ ID NO: 359) Tiling 70 nt 54gatagaatgttctaaacCatcctgcggcct Has a 5′ G for U6 50C mismatchctactctgcattcaattacatactgacacatt expression distancecggcaacat (SEQ ID NO: 360) Tiling 70 nt 52 gccatagaatgttctaaacCatcctgcggHas a 5′ G for U6 50C mismatch cctctactctgcattcaattacatactgacacexpression distance attcggcaac (SEQ ID NO: 361) Tiling 70 nt 50gttccatagaatgttctaaacCatcctgcg Has a 5′ G for U6 50C mismatchgcctctactctgcattcaattacatactgac expression distanceacattcggca (SEQ ID NO: 362) Tiling 70 nt 48gctttccatagaatgttctaaacCatcctgc Has a 5′ G for U6 50C mismatchggcctctactctgcattcaattacatactga expression distancecacattcgg (SEQ ID NO: 363) Tiling 70 nt 46gctctttccatagaatgttctaaacCatcct Has a 5′ G for U6 50C mismatchgcggcctctactctgcattcaattacatact expression distancegacacattc (SEQ ID NO: 364) Tiling 70 nt 44gatctctttccatagaatgttctaaacCatc Has a 5′ G for U6 50C mismatchctgcggcctctactctgcattcaattacata expression distancectgacacat (SEQ ID NO: 365) Tiling 70 nt 42ggaatctctttccatagaatgttctaaacCa Has a 5′ G for U6 50C mismatchtcctgcggcctctactctgcattcaattacat expression distanceactgacac (SEQ ID NO: 366) Tiling 70 nt 40gtggaatctctttccatagaatgttctaaac Has a 5′ G for U6 50C mismatchCatcctgcggcctctactctgcattcaatta expression distancecatactgac (SEQ ID NO: 367) Tiling 70 nt 38gactggaatctctttccatagaatgttctaaa Has a 5′ G for U6 50C mismatchcCatcctgcggcctctactctgcattcaatt expression distanceacatactg (SEQ ID NO: 368) Tiling 70 nt 36ggaactggaatctctttccatagaatgttct Has a 5′ G for U6 50C mismatchaaacCatcctgcggcctctactctgcattc expression distanceaattacatac (SEQ ID NO: 369) Tiling 70 nt 34gtggaactggaatctctttccatagaatgtt Has a 5′ G for U6 50C mismatchctaaacCatcctgcggcctctactctgcat expression distancetcaattacat (SEQ ID NO: 370) Tiling 70 nt 32gcctggaactggaatctctttccatagaatg Has a 5′ G for U6 50C mismatchttctaaacCatcctgcggcctctactctgc expression distanceattcaattac (SEQ ID NO: 371) Tiling 70 nt 30gttcctggaactggaatctctttccatagaa Has a 5′ G for U6 50C mismatchtgttctaaacCatcctgcggcctctactctg expression distancecattcaatt (SEQ ID NO: 372) Tiling 70 nt 28gggttcctggaactggaatctctttccatag Has a 5′ G for U6 50C mismatchaatgttctaaacCatcctgcggcctctact expression distancectgcattcaa (SEQ ID NO: 373) Tiling 70 nt 26gcaggttcctggaactggaatctctttccat Has a 5′ G for U6 50C mismatchagaatgttctaaacCatcctgcggcctcta expression distancectctgcattc (SEQ ID NO: 374) Tiling 70 nt 24gaccaggttcctggaactggaatctctttcc Has a 5′ G for U6 50C mismatchatagaatgttctaaacCatcctgcggcctc expression distancetactctgcat (SEQ ID NO: 375) Tiling 70 nt 22ggtaccaggttcctggaactggaatctcttt Has a 5′ G for U6 50C mismatchccatagaatgttctaaacCatcctgcggcc expression distancetctactctgc (SEQ ID NO: 376) Tiling 70 nt 20gatgtaccaggttcctggaactggaatctc Has a 5′ G for U6 50C mismatchtttccatagaatgttctaaacCatcctgcgg expression distancecctctactct (SEQ ID NO: 377) Tiling 70 nt 18ggtatgtaccaggttcctggaactggaatc Has a 5′ G for U6 50C mismatchtctttccatagaatgttctaaacCatcctgc expression distanceggcctctact (SEQ ID NO: 378) Tiling 70 nt 16gacgtatgtaccaggttcctggaactggaa Has a 5′ G for U6 50C mismatchtctctttccatagaatgttctaaacCatcctg expression distancecggcctcta (SEQ ID NO: 379) Tiling 70 nt 14gacacgtatgtaccaggttcctggaactgg Has a 5′ G for U6 50C mismatchaatctctttccatagaatgttctaaacCatc expression distancectgcggcctc (SEQ ID NO: 380) Tiling 70 nt 12gcaacacgtatgtaccaggttcctggaact Has a 5′ G for U6 50C mismatchggaatctctttccatagaatgttctaaacCa expression distancetcctgcggcc (SEQ ID NO: 381) Tiling 70 nt 10gcccaacacgtatgtaccaggttcctgga Has a 5′ G for U6 50C mismatchactggaatctctttccatagaatgttctaaac expression distanceCatcctgcgg (SEQ ID NO: 382) Tiling 70 nt 8 ggacccaacacgtatgtaccaggttcctgHas a 5′ G for U6 50C mismatch gaactggaatctctttccatagaatgttctaaexpression distance acCatcctgc (SEQ ID NO: 383) Tiling 70 nt 6gttgacccaacacgtatgtaccaggttcct Has a 5′ G for U6 50C mismatchggaactggaatctctttccatagaatgttct expression distanceaaacCatcct (SEQ ID NO: 384) Tiling 70 nt 4gccttgacccaacacgtatgtaccaggttc Has a 5′ G for U6 50C mismatchctggaactggaatctctttccatagaatgtt expression distancectaaacCatc (SEQ ID NO: 385) Tiling 70 nt 2gttccttgacccaacacgtatgtaccaggtt Has a 5′ G for U6 50C mismatchcctggaactggaatctctttccatagaatgt expression distancetctaaacCa (SEQ ID NO: 386) Tiling 84 nt 84gCatcctgcggcctctactctgcattcaatt Has a 5′ G for U6 50C mismatchacatactgacacattcggcaacatgtttttc expression distancectggtttattttcacacagtcca (SEQ ID NO: 387) Tiling 84 nt 82gacCatcctgcggcctctactctgcattca Has a 5′ G for U6 50C mismatchattacatactgacacattcggcaacatgtttt expression distancetcctggtttattttcacacagtc (SEQ ID NO: 388) Tiling 84 nt 80gaaacCatcctgcggcctctactctgcatt Has a 5′ G for U6 50C mismatchcaattacatactgacacattcggcaacatgt expression distancettttcctggtttattttcacacag (SEQ ID NO: 389) Tiling 84 nt 78gctaaacCatcctgcggcctctactctgca Has a 5′ G for U6 50C mismatchttcaattacatactgacacattcggcaacat expression distancegtttttcctggtttattttcacac (SEQ ID NO: 390) Tiling 84 nt 76gttctaaacCatcctgcggcctctactctg Has a 5′ G for U6 50C mismatchcattcaattacatactgacacattcggcaac expression distanceatgtttttcctggtttattttcac (SEQ ID NO: 391) Tiling 84 nt 74gtgttctaaacCatcctgcggcctctactct Has a 5′ G for U6 50C mismatchgcattcaattacatactgacacattcggcaa expression distancecatgtttttcctggtttattttc (SEQ ID NO: 392) Tiling 84 nt 72gaatgttctaaacCatcctgcggcctctac Has a 5′ G for U6 50C mismatchtctgcattcaattacatactgacacattcgg expression distancecaacatgtttttcctggtttattt (SEQ ID NO: 393) Tiling 84 nt 70gagaatgttctaaacCatcctgcggcctct Has a 5′ G for U6 50C mismatchactctgcattcaattacatactgacacattc expression distanceggcaacatgtttttcctggtttat (SEQ ID NO: 394) Tiling 84 nt 68gatagaatgttctaaacCatcctgcggcct Has a 5′ G for U6 50C mismatchctactctgcattcaattacatactgacacatt expression distancecggcaacatgtttttcctggttt (SEQ ID NO: 395) Tiling 84 nt 66gccatagaatgttctaaacCatcctgcgg Has a 5′ G for U6 50C mismatchcctctactctgcattcaattacatactgacac expression distanceattcggcaacatgtttttcctggt (SEQ ID NO: 396) Tiling 84 nt 64gttccatagaatgttctaaacCatcctgcg Has a 5′ G for U6 50C mismatchgcctctactctgcattcaattacatactgac expression distanceacattcggcaacatgtttttcctg (SEQ ID NO: 397) Tiling 84 nt 62gctttccatagaatgttctaaacCatcctgc Has a 5′ G for U6 50C mismatchggcctctactctgcattcaattacatactga expression distancecacattcggcaacatgtttttcc (SEQ ID NO: 398) Tiling 84 nt 60gctctttccatagaatgttctaaacCatcct Has a 5′ G for U6 50C mismatchgcggcctctactctgcattcaattacatact expression distancegacacattcggcaacatgttttt (SEQ ID NO: 399) Tiling 84 nt 58gatctctttccatagaatgttctaaacCatc Has a 5′ G for U6 50C mismatchctgcggcctctactctgcattcaattacata expression distancectgacacattcggcaacatgttt (SEQ ID NO: 400) Tiling 84 nt 56ggaatctctttccatagaatgttctaaacCa Has a 5′ G for U6 50C mismatchtcctgcggcctctactctgcattcaattacat expression distanceactgacacattcggcaacatgt (SEQ ID NO: 401) Tiling 84 nt 54gtggaatctctttccatagaatgttctaaac Has a 5′ G for U6 50C mismatchCatcctgcggcctctactctgcattcaatta expression distancecatactgacacattcggcaacat (SEQ ID NO: 402) Tiling 84 nt 52gactggaatctctttccatagaatgttctaaa Has a 5′ G for U6 50C mismatchcCatcctgcggcctctactctgcattcaatt expression distanceacatactgacacattcggcaac (SEQ ID NO: 403) Tiling 84 nt 50ggaactggaatctctttccatagaatgttct Has a 5′ G for U6 50C mismatchaaacCatcctgcggcctctactctgcattc expression distanceaattacatactgacacattcggca (SEQ ID NO: 404) Tiling 84 nt 48gtggaactggaatctctttccatagaatgtt Has a 5′ G for U6 50C mismatchctaaacCatcctgcggcctctactctgcat expression distancetcaattacatactgacacattcgg (SEQ ID NO: 405) Tiling 84 nt 46gcctggaactggaatctctttccatagaatg Has a 5′ G for U6 50C mismatchttctaaacCatcctgcggcctctactctgc expression distanceattcaattacatactgacacattc (SEQ ID NO: 406) Tiling 84 nt 44gttcctggaactggaatctctttccatagaa Has a 5′ G for U6 50C mismatchtgttctaaacCatcctgcggcctctactctg expression distancecattcaattacatactgacacat (SEQ ID NO: 407) Tiling 84 nt 42gggttcctggaactggaatctctttccatag Has a 5′ G for U6 50C mismatchaatgttctaaacCatcctgcggcctctact expression distancectgcattcaattacatactgacac (SEQ ID NO: 408) Tiling 84 nt 40gcaggttcctggaactggaatctctttccat Has a 5′ G for U6 50C mismatchagaatgttctaaacCatcctgcggcctcta expression distancectctgcattcaattacatactgac (SEQ ID NO: 409) Tiling 84 nt 38gaccaggttcctggaactggaatctctttcc Has a 5′ G for U6 50C mismatchatagaatgttctaaacCatcctgcggcctc expression distancetactctgcattcaattacatactg (SEQ ID NO: 410) Tiling 84 nt 36ggtaccaggttcctggaactggaatctcttt Has a 5′ G for U6 50C mismatchccatagaatgttctaaacCatcctgcggcc expression distancetctactctgcattcaattacatac (SEQ ID NO: 411) Tiling 84 nt 34gatgtaccaggttcctggaactggaatctc Has a 5′ G for U6 50C mismatchtttccatagaatgttctaaacCatcctgcgg expression distancecctctactctgcattcaattacat (SEQ ID NO: 412) Tiling 84 nt 32ggtatgtaccaggttcctggaactggaatc Has a 5′ G for U6 50C mismatchtctttccatagaatgttctaaacCatcctgc expression distanceggcctctactctgcattcaattac (SEQ ID NO: 413) Tiling 84 nt 30gacgtatgtaccaggttcctggaactggaa Has a 5′ G for U6 50C mismatchtctctttccatagaatgttctaaacCatcctg expression distancecggcctctactctgcattcaatt (SEQ ID NO: 414) Tiling 84 nt 28gacacgtatgtaccaggttcctggaactgg Has a 5′ G for U6 50C mismatchaatctctttccatagaatgttctaaacCatc expression distancectgcggcctctactctgcattcaa (SEQ ID NO: 415) Tiling 84 nt 26gcaacacgtatgtaccaggttcctggaact Has a 5′ G for U6 50C mismatchggaatctctttccatagaatgttctaaacCa expression distancetcctgcggcctctactctgcattc (SEQ ID NO: 416) Tiling 84 nt 24gcccaacacgtatgtaccaggttcctgga Has a 5′ G for U6 50C mismatchactggaatctctttccatagaatgttctaaac expression distanceCatcctgcggcctctactctgcat (SEQ ID NO: 417) Tiling 84 nt 22ggacccaacacgtatgtaccaggttcctg Has a 5′ G for U6 50C mismatchgaactggaatctctttccatagaatgttctaa expression distanceacCatcctgcggcctctactctgc (SEQ ID NO: 418) Tiling 84 nt 20gttgacccaacacgtatgtaccaggttcct Has a 5′ G for U6 50C mismatchggaactggaatctctttccatagaatgttct expression distanceaaacCatcctgcggcctctactct (SEQ ID NO: 419) Tiling 84 nt 18gccttgacccaacacgtatgtaccaggttc Has a 5′ G for U6 50C mismatchctggaactggaatctctttccatagaatgtt expression distancectaaacCatcctgcggcctctact (SEQ ID NO: 420) Tiling 84 nt 16gttccttgacccaacacgtatgtaccaggtt Has a 5′ G for U6 50C mismatchcctggaactggaatctctttccatagaatgt expression distancetctaaacCatcctgcggcctcta (SEQ ID NO: 421) Tiling 84 nt 14gggttccttgacccaacacgtatgtaccag Has a 5′ G for U6 50C mismatchgttcctggaactggaatctctttccatagaa expression distancetgttctaaacCatcctgcggcctc (SEQ ID NO: 422) Tiling 84 nt 12gttggttccttgacccaacacgtatgtacca Has a 5′ G for U6 50C mismatchggttcctggaactggaatctctttccataga expression distanceatgttctaaacCatcctgcggcc (SEQ ID NO: 423) Tiling 84 nt 10gccttggttccttgacccaacacgtatgtac Has a 5′ G for U6 50C mismatchcaggttcctggaactggaatctctttccata expression distancegaatgttctaaacCatcctgcgg (SEQ ID NO: 424) Tiling 84 nt 8ggcccttggttccttgacccaacacgtatgt Has a 5′ G for U6 50C mismatchaccaggttcctggaactggaatctctttcca expression distancetagaatgttctaaacCatcctgc (SEQ ID NO: 425) Tiling 84 nt 6gccgcccttggttccttgacccaacacgta Has a 5′ G for U6 50C mismatchtgtaccaggttcctggaactggaatctcttt expression distanceccatagaatgttctaaacCatcct (SEQ ID NO: 426) Tiling 84 nt 4gcgccgcccttggttccttgacccaacac Has a 5′ G for U6 50C mismatchgtatgtaccaggttcctggaactggaatct expression distancectttccatagaatgttctaaacCatc (SEQ ID NO: 427) Tiling 84 nt 2ggtcgccgcccttggttccttgacccaaca Has a 5′ G for U6 50C mismatchcgtatgtaccaggttcctggaactggaatc expression distancetctttccatagaatgttctaaacCa (SEQ ID NO: 428 ADAR non- GTAATGCCTGGCTTGTCGHas a 5′ G for U6 50C targeting guide ACGCATAGTCTG (SEQ ID expressionNO: 429) PFS binding gaaaacgcaggttcctcCagtttcgggag Has a 5′ G for U6 51Bscreen guide for cagcgcacgtctccctgtagtc (SEQ expression TAG motifID NO: 430) PFS binding gacgcaggttcctctagCttcgggagcag Has a 5′ G for U651B screen guide for cgcacgtctccctgtagtcaag (SEQ expression AAC motifID NO: 431) PFS binding GTAATGCCTGGCTTGTCG Has a 5′ G for U6 51Bscreen non- ACGCATAGTCTG (SEQ ID expression targeting NO: 432)Motif preference gatagaatgttctaaacCatcctgcggcct Has a 5′ G for U6 51Ctargeting guide ctactctgcattcaattacat expression Motif preferenceGTAATGCCTGGCTTGTCG Has a 5′ G for U6 51C non-targetingACGCATAGTCTG (SEQ ID expression guide NO: 433) PPIB tiling guidegCaaggccacaaaattatccactgtttttg Has a 5′ G for U6 57D 50 mismatchgaacagtctttccgaagagac (SEQ expression distance ID NO: 435)PPIB tiling guide gcctgtagcCaaggccacaaaattatcca Has a 5′ G for U6 57D42 mismatch ctgtttttggaacagtctttcc (SEQ ID expression distance NO: 436)PPIB tiling guide gctttctctcctgtagcCaaggccacaaaa Has a 5′ G for U6 57D34 mismatch ttatccactgtttttggaaca (SEQ ID expression distance NO: 437)PPIB tiling guide ggccaaatcctttctctcctgtagcCaagg Has a 5′ G for U6 57D26 mismatch ccacaaaattatccactgttt (SEQ ID expression distance NO: 438)PPIB tiling guide gtttttgtagccaaatcctttctctcctgtagc Has a 5′ G for U657D 18 mismatch Caaggccacaaaattatc (SEQ ID expression distance NO: 439)PPIB tiling guide gatttgctgtttttgtagccaaatcctttctct Has a 5′ G for U657D 10 mismatch cctgtagcCaaggccaca (SEQ ID expression distance NO: 440)PPIB tiling guide gacgatggaatttgctgtttttgtagccaaat Has a 5′ G for U6 57D2 mismatch cctttctctcctgtagcCa (SEQ ID expression distance NO: 441)Targeting guide, gatagaatgttctaaacGatcctgcggcct Has a 5′ G for U6 57Dopposite base G ctactctgcattcaattacat (SEQ ID expression NO: 442)Targeting guide, gatagaatgttctaaacAatcctgcggcct Has a 5′ G for U6 57Dopposite base A ctactctgcattcaattacat (SEQ ID expression NO: 443)Targeting guide, gatagaatgttctaaacTatcctgcggcct Has a 5′ G for U6 57Dopposite base C ctactctgcattcaattacat (SEQ ID expression NO: 444)AVPR2 guide 37 ggtcccacgcggccCacagctgcacca Has a 5′ G for U6 52Amismatch ggaagaagggtgcccagcacagca expression distance (SEQ ID NO: 445)AVPR2 guide 35 ggggtcccacgcggccCacagctgcac Has a 5′ G for U6 52Amismatch caggaagaagggtgcccagcacag expression distance (SEQ ID NO: 446)AVPR2 guide 33 gccgggtcccacgcggccCacagctgc Has a 5′ G for U6 52Amismatch accaggaagaagggtgcccagcac expression distance (SEQ ID NO: 447)FANCC guide 37 gggtgatgacatccCaggcgatcgtgtg Has a 5′ G for U6 52Bmismatch gcctccaggagcccagagcagga expression distance (SEQ ID NO: 448)FANCC guide 35 gagggtgatgacatccCaggcgatcgtg Has a 5′ G for U6 52Bmismatch tggcctccaggagcccagagcag expression distance (SEQ ID NO: 449)FANCC guide 32 gatcagggtgatgacatccCaggcgatc Has a 5′ G for U6 52Bmismatch gtgtggcctccaggagcccagag expression distance (SEQ ID NO: 450)Synthetic disease ggtggctccattcactcCaatgctgagca Has a 5′ G for U6 52Egene target cttccacagagtgggttaaagc (SEQ expression IL2RG ID NO: 451)Synthetic disease gtttctaatatattttgCcagactgatggact Has a 5′ G for U6 52Egene target F8 attctcaattaataatgat (SEQ ID expression NO: 452)Synthetic disease gagatgttgctgtggatCcagtccacagc Has a 5′ G for U6 52Egene target LDLR cagcccgtcgggggcctggatg (SEQ expression ID NO: 453)Synthetic disease gcaggccggcccagctgCcaggtgcac Has a 5′ G for U6 52Egene target CBS ctgctcggagcatcgggccggatc expression (SEQ ID NO: 454)Synthetic disease gcaaagaacctctgggtCcaagggtaga Has a 5′ G for U6 52Egene target HBB ccaccagcagcctgcccagggcc expression (SEQ ID NO: 455)Synthetic disease gaagagaaacttagtttCcagggctttggt Has a 5′ G for U6 52Egene target agagggcaaaggttgatagca (SEQ expression ALDOB ID NO: 456)Synthetic disease gtcagcctagtgcagagCcactggtagtt Has a 5′ G for U6 52Egene target DMD ggtggttagagtttcaagttcc (SEQ ID expression NO: 457)Synthetic disease ggctcattgtgaacaggCcagtaatgtcc Has a 5′ G for U6 52Egene target gggatggggcggcataggcggg expression SMAD4 (SEQ ID NO: 458)Synthetic disease gtagctaaagaacttgaCcaagacatatc Has a 5′ G for U6 52Egene target aggatccacctcagctcctaga (SEQ expression BRCA2 ID NO: 459)Synthetic disease ggggcattgttctgtgcCcagtcctgctgg Has a 5′ G for U6 52Egene target tagacctgctccccggtggct (SEQ ID expression GRIN2A NO: 460)Synthetic disease gagaagtcgttcatgtgCcaccgtggga Has a 5′ G for U6 52Egene target gcgtacagtcatcattgatcttg (SEQ expression SCN9A ID NO: 461)Synthetic disease gggattaatgctgaacgCaccaaagttca Has a 5′ G for U6 52Egene target tcccaccacccatattactacc (SEQ expression TARDBP ID NO: 462)Synthetic disease gctccaaaggctttcctCcactgttgcaaa Has a 5′ G for U6 52Egene target CFTR gttattgaatcccaagacaca (SEQ ID expression NO: 463)Synthetic disease gatgaatgaacgatttcCcagaactcccta Has a 5′ G for U6 52Egene target atcagaacagagtccctggta (SEQ expression UBE3A ID NO: 464)Synthetic disease ggagcctctgccggagcCcagagaacc Has a 5′ G for U6 52Egene target cgagagtcagacagagccagcgcc expression SMPD1 (SEQ ID NO: 465)Synthetic disease ggcttccgtggagacacCcaatcaatttg Has a 5′ G for U6 52Egene target aagagatcttgaagtgatgcca (SEQ expression USH2A ID NO: 466)Synthetic disease gtgggactgccctcctcCcatttgcagatg Has a 5′ G for U6 52Egene target ccgtcgtagaatcgcagcagg (SEQ expression MEN1 ID NO: 467)Synthetic disease gcttcttcaatagttctCcagctacactggc Has a 5′ G for U6 52Egene target aggcatatgcccgtgttcct (SEQ ID expression C8orf37 NO: 468)Synthetic disease gattccttttcttcgtcCcaattcacctcagt Has a 5′ G for U6 52Egene target ggctagtcgaagaatgaag (SEQ ID expression MLH1 NO: 469)Synthetic disease gcagcttcagcaccttcCagtcagactcct Has a 5′ G for U6 52Egene target TSC2 gcttcaagcactgcagcagga (SEQ expression ID NO: 470)Synthetic disease gccatttgcttgcagtgCcactccagagg Has a 5′ G for U6 52Egene target NF1 attccggattgccataaatact (SEQ ID expression NO: 471)Synthetic disease gttcaatagttttggtcCagtatcgtttacag Has a 5′ G for U6 52Egene target MSH6 cccttcttggtagatttca (SEQ ID expression NO: 472)Synthetic disease ggcaaccgtcttctgacCaaatggcagaa Has a 5′ G for U6 52Egene target SMN1 catttgtccccaactttccact (SEQ ID expression NO: 473)Synthetic disease gcgactttccaatgaacCactgaagccca Has a 5′ G for U6 52Egene target ggtatgacaaagccgatgatct (SEQ expression SH3TC2 ID NO: 474)Synthetic disease gtttacactcatgcttcCacagctttaacag Has a 5′ G for U6 52Egene target atcatttggttccttgatga (SEQ ID expression DNAH5 NO: 475)Synthetic disease gcttaagcttccgtgtcCagccttcaggca Has a 5′ G for U6 52Egene target gggtggggtcatcatacatgg (SEQ expression MECP2 ID NO: 476)Synthetic disease ggacagctgggctgatcCatgatgtcatc Has a 5′ G for U6 52Egene target cagaaacactggggaccctcag (SEQ expression ADGRV1 ID NO: 477)Synthetic disease gtctcatctcaactttcCatatccgtatcatg Has a 5′ G for U6 52Egene target AHI1 gaatcatagcatcctgtaa (SEQ ID expression NO: 478)Synthetic disease gcatgcagacgcggttcCactcgcagcc Has a 5′ G for U6 52Egene target PRKN acagttccagcaccactcgagcc (SEQ expression ID NO: 479)Synthetic disease gttggttagggtcaaccCagtattctccac Has a 5′ G for U6 52Egene target tcttgagttcaggatggcaga (SEQ ID expression COL3A1 NO: 480)Synthetic disease gctacactgtccaacacCcactctcgggt Has a 5′ G for U6 52Egene target caccacaggtgcctcacacatc (SEQ expression BRCA1 ID NO: 481)Synthetic disease gctgcactgtgtaccccCagagctccgtg Has a 5′ G for U6 52Egene target ttgccgacatcctggggtggct (SEQ expression MYBPC3 ID NO: 482)Synthetic disease gagcttcctgccactccCaacaggtttcac Has a 5′ G for U6 52Egene target APC agtaagcgcgtatctgttcca (SEQ ID expression NO: 483)Synthetic disease gacggcaagagcttaccCagtcacttgtg Has a 5′ G for U6 52Egene target tggagacttaaatacttgcata (SEQ ID expression BMPR2 NO: 484)KRAS tiling gCaaggccacaaaattatccactgtttttg Has a 5′ G for U6 53Aguide 50 gaacagtctttccgaagagac (SEQ expression mismatch ID NO: 485)distance KRAS tiling gcctgtagcCaaggccacaaaattatcca Has a 5′ G for U6 53Aguide 42 ctgtttttggaacagtctttcc (SEQ ID expression mismatch NO: 486)distance KRAS tiling gctttctctcctgtagcCaaggccacaaaa Has a 5′ G for U653A guide 34 ttatccactgtttttggaaca (SEQ ID expression mismatch NO: 487)distance KRAS tiling ggccaaatcctttctctcctgtagcCaagg Has a 5′ G for U653A guide 26 ccacaaaattatccactgttt (SEQ ID expression mismatch NO: 488)distance KRAS tiling gtttttgtagccaaatcctttctctcctgtagc Has a 5′ G for U653A guide 18 Caaggccacaaaattatc (SEQ ID expression mismatch NO: 489)distance KRAS tiling gatttgctgtttttgtagccaaatcctttctct Has a 5′ G for U653A guide 10 cctgtagcCaaggccaca (SEQ ID expression mismatch NO: 490)distance KRAS tiling gacgatggaatttgctgtttttgtagccaaat Has a 5′ G for U653A guide 2 mismatch cctttctctcctgtagcCa (SEQ ID expression distanceNO: 491) KRAS tiling non- GTAATGCCTGGCTTGTCG Has a 5′ G for U6 53Atargeting guide ACGCATAGTCTG (SEQ ID expression NO: 492) Luciferase W85XgatagaatgttctaaacCatcctgcggcct Has a 5′ G for U6 53B targeting guidectactctgcattcaattacat (SEQ ID expression for transcriptome NO: 493)specificity Non-targeting GCAGGGTTTTCCCAGTCA Has a 5′ G for U6 53Cguide for CGACGTTGTAAAGTTG expression transcriptome (SEQ ID NO: 494)specificity endogenous gtcaaggcactcttgccCacgccaccag Has a 5′ G for U654F KRAS guide 2 ctccaactaccacaagtttatat (SEQ expression ID NO: 495)endogenous PPIB gcaaagatcacccggccCacatcttcatc Has a 5′ G for U6 54Gguide 1 tccaattcgtaggtcaaaatac (SEQ expression ID NO: 496) endogenousGcgccaccagctccaacCaccacaagtt Has a 5′ G for U6 54F KRAS guide 1tatattcagtcattttcagcagg (SEQ expression ID NO: 497) endogenousGtttctccatcaattacCacttgcttcctgta Has a 5′ G for U6 54F KRAS guide 3ggaatcctctattGTtgga (SEQ ID expression NO: 498) endogenous PPIBGctttctctcctgtagcCaaggccacaaa Has a 5′ G for U6 54G guide 2attatccactgtttttggaaca (SEQ ID expression NO: 499) endogenous non-GTAATGCCTGGCTTGTCG Has a 5′ G for U6 54F targeting guideACGCATAGTCTG (SEQ ID expression NO: 500) BoxB Cluc guidetctttccataGGCCCTGAAAAA Has a 5′ G for U6 62B GGGCCtgttctaaacCatcctgcggcexpression ctctactcGGCCCTGAAAAAG GGCCattcaattac (SEQ ID NO: 501)BoxB non- cagctggcgaGGCCCTGAAAA Has a 5′ G for U6 62B targeting guideAGGGCCggggatgtgcCgcaaggc expression gattaagttggGGCCCTGAAAAAGGGCCacgccagggt (SEQ ID NO: 502) Stafforst full GTGGAATAGTATAACAATHas a 5′ G for U6 62C length ADAR2 ATGCTAAATGTTGTTATA expression guide 1GTATCCCACtctaaaCCAtcctg cgGGGCCCTCTTCAGGGCC C (SEQ ID NO: 503)Stafforst full GTGGAATAGTATAACAAT Has a 5′ G for U6 62C length ADAR2ATGCTAAATGTTGTTATA expression non-targeting GTATCCCACaccctggcgttacccguide aGGGCCCTCTTCAGGGCCC (SEQ ID NO: 504)

REFERENCES

-   1. P. D. Hsu, E. S. Lander, F. Zhang, Development and applications    of CRISPR-Cas9 for genome engineering. Cell 157, 1262-1278 (2014).-   2. A. C. Komor, A. H. Badran, D. R. Liu, CRISPR-Based Technologies    for the Manipulation of Eukaryotic Genomes. Cell 168, 20-36 (2017).-   3. L. Cong et al., Multiplex genome engineering using CRISPR/Cas    systems. Science 339, 819-823 (2013).-   4. P. Mali et al., RNA-guided human genome engineering via Cas9.    Science 339, 823 826 (2013).-   5. B. Zetsche et al., Cpf1 is a single RNA-guided endonuclease of a    class 2 CRISPR-Cas system. Cell 163, 759-771 (2015).-   6. H. Kim, J. S. Kim, A guide to genome engineering with    programmable nucleases. Nat Rev Genet 15, 321-334 (2014).-   7. A. C. Komor, Y. B. Kim, M. S. Packer, J. A. Zuris, D. R. Liu,    Programmable editing of a target base in genomic DNA without    double-stranded DNA cleavage. Nature 533, 420-424 (2016).-   8. K. Nishida et al., Targeted nucleotide editing using hybrid    prokaryotic and vertebrate adaptive immune systems. Science 353,    (2016).-   9. Y. B. Kim et al., Increasing the genome-targeting scope and    precision of base editing with engineered Cas9-cytidine deaminase    fusions. Nat Biotechnol 35, 371-376 (2017).-   10. O. O. Abudayyeh et al., C2c2 is a single-component programmable    RNA-guided RNA-targeting CRISPR effector. Science 353, aaf5573    (2016).-   11. S. Shmakov et al., Discovery and Functional Characterization of    Diverse Class 2 CRISPR-Cas Systems. Mol Cell 60, 385-397 (2015).-   12. S. Shmakov et al., Diversity and evolution of class 2 CRISPR-Cas    systems. Nat Rev Microbiol 15, 169-182 (2017).-   13. A. A. Smargon et al., Cas13b Is a Type VI-B CRISPR-Associated    RNA-Guided RNase Differentially Regulated by Accessory Proteins    Csx27 and Csx28. Mol Cell 65, 618-630 e617 (2017).-   14. J. S. Gootenberg et al., Nucleic acid detection with    CRISPR-Cas13a/C2c2. Science 356, 438-442 (2017).-   15. O. O. Abudayyeh et al., RNA targeting with CRISPR-Cas13a. Nature    in press, (2017).-   16. K. Nishikura, Functions and regulation of RNA editing by ADAR    deaminases. Annu Rev Biochem 79, 321-349 (2010).-   17. B. L. Bass, H. Weintraub, An unwinding activity that covalently    modifies its double-stranded RNA substrate. Cell 55, 1089-1098    (1988).-   18. M. M. Matthews et al., Structures of human ADAR2 bound to dsRNA    reveal base-flipping mechanism and basis for site selectivity. Nat    Struct Mol Biol 23, 426-433 (2016).-   19. A. Kuttan, B. L. Bass, Mechanistic insights into editing-site    specificity of ADARs. Proc Natl Acad Sci USA 109, E3295-3304 (2012).-   20. S. K. Wong, S. Sato, D. W. Lazinski, Substrate recognition by    ADAR1 and ADAR2. RNA 7, 846-858 (2001).-   21. M. Fukuda et al., Construction of a guide-RNA for site-directed    RNA mutagenesis utilising intracellular A-to-I RNA editing. Sci Rep    7, 41478 (2017).-   22. M. F. Montiel-Gonzalez, I. Vallecillo-Viejo, G. A.    Yudowski, J. J. Rosenthal, Correction of mutations within the cystic    fibrosis transmembrane conductance regulator by site-directed RNA    editing. Proc Natl Acad Sci USA 110, 18285-18290 (2013).-   23. M. F. Montiel-Gonzalez, I. C. Vallecillo-Viejo, J. J. Rosenthal,    An efficient system for selectively altering genetic information    within mRNAs. Nucleic Acids Res 44, e157 (2016).-   24. J. Wettengel, P. Reautschnig, S. Geisler, P. J. Kahle, T.    Stafforst, Harnessing human ADAR2 for RNA repair—Recoding a PINK1    mutation rescues mitophagy. Nucleic Acids Res 45, 2797-2808 (2017).-   25. Y. Wang, J. Havel, P. A. Beal, A Phenotypic Screen for    Functional Mutants of Human Adenosine Deaminase Acting on RNA 1. ACS    Chem Biol 10, 2512-2519 (2015).-   26. K. A. Lehmann, B. L. Bass, Double-stranded RNA adenosine    deaminases ADAR1 and ADAR2 have overlapping specificities.    Biochemistry 39, 12875-12884 (2000).-   27. Y. Zheng, C. Lorenzo, P. A. Beal, DNA editing in DNA/RNA hybrids    by adenosine deaminases that act on RNA. Nucleic Acids Res 45,    3369-3377 (2017).-   28. K. Gao et al., A de novo loss-of-function GRIN2A mutation    associated with childhood focal epilepsy and acquired epileptic    aphasia. PLoS One 12, e0170818 (2017).-   29. H. M. Lanoiselee et al., APP, PSEN1, and PSEN2 mutations in    early-onset Alzheimer disease: A genetic screening study of familial    and sporadic cases. PLoS Med 14, e1002270 (2017).-   30. C. Ballatore, V. M. Lee, J. Q. Trojanowski, Tau-mediated    neurodegeneration in Alzheimer's disease and related disorders. Nat    Rev Neurosci 8, 663-672 (2007).-   31. Y. Li et al., Carriers of rare missense variants in IFIH1 are    protected from psoriasis. J Invest Dermatol 130, 2768-2772 (2010).-   32. R. S. Finkel et al., Treatment of infantile-onset spinal    muscular atrophy with nusinersen: a phase 2, open-label,    dose-escalation study. Lancet 388, 3017-3026 (2016).

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

1-50. (canceled)
 51. A non-naturally occurring or engineered compositionfor modifying a target RNA sequence, said composition comprising: (a) aCas13b effector protein comprising any one of amino acid sequences ofSEQ ID NO: 31-38, (b) an engineered guide molecule, and (c) one or moreheterologous functional domains associated with the Cas13b effectorprotein, wherein the engineered guide molecule is capable of forming aCRISPR-Cas complex with the Cas13b effector protein and comprises (i) aguide sequence that directs sequence-specific binding to a target RNAsequence other than the naturally occurring Cas13b protospacer and thatreprograms the CRISPR-Cas complex to bind said target RNA sequence, and(b) a direct repeat sequence, and wherein the one or more heterologousfunctional domains modifies the target RNA sequence.
 52. The compositionof claim 51, wherein the Cas13b effector protein is catalyticallyinactive.
 53. The composition of claim 51, wherein the Cas13b effectorprotein comprises one or more mutations to the two HEPN domains.
 54. Thecomposition of claim 51, wherein the Cas13b effector protein comprises amutation in one or more of positions R116A, H121A, R1177A, and H1182A ofCas13b effector protein originating from Bergeyella zoohelcum ATCC 43767or amino acid positions corresponding thereto of a Cas13b ortholog. 55.The composition of claim 51, wherein the Cas13b effector protein istruncated.
 56. The composition of claim 55, wherein the Cas13b effectorprotein is a truncated functional variant of the corresponding wildtypeCas13b.
 57. The composition of claim 56, wherein the Cas13b effectorprotein corresponds to nucleotides 1-984 of Prevotella sp. P5-125Cas13b.
 58. The composition of claim 55, wherein the Cas13b effectorprotein is catalytically inactive.
 59. The composition of claim 51,wherein the guide sequence hybridizes to a target RNA sequencecomprising an adenine to form an RNA duplex, wherein the guide sequencecomprises a non-pairing cytosine at a position corresponding to saidadenine, resulting in an A-C mismatch in the RNA duplex formed.
 60. Thecomposition of claim 51, wherein the guide sequence comprises more thanone mismatch corresponding to different adenosine sites in the targetRNA sequence or wherein two guide molecules are used, each comprising amismatch corresponding to a different adenosine site in the target RNAsequence.
 61. The composition of claim 51, wherein the one or moreheterologous functional domains modifies the target RNA sequence byconverting adenosine to inosine.
 62. The composition of claim 61,wherein the one or more heterologous functional domains comprises anadenosine deaminase.
 63. The composition of claim 62, wherein theadenosine deaminase is fused to a N- or C-terminus of the Cas 13beffector protein.
 64. The composition of claim 63, wherein the adenosinedeaminase is fused by a linker.
 65. The composition of claim 64, whereinthe linker is (GGGGS)₃₋₁₁, GSG₅, or LEPGEKPYKCPECGKSFSQSGALTRHQRTHTR, orwherein the linker is an XTEN linker.
 66. The composition of claim 62,wherein the adenosine deaminase is linked to an adaptor protein and theguide molecule.
 67. The composition of claim 62, wherein thecatalytically inactive Cas13b effector protein comprises an aptamersequence capable of binding to an adaptor protein, wherein the adaptorprotein is selected from MS2, PP7, Qβ, F2, GA, fr, JP501, M12, R17,BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19,AP205, ϕCb5, ϕCb8r, ϕCb12r, ϕCb23r, 7s, and PRR1.
 68. The composition ofclaim 62, wherein the adenosine deaminase is an RNA-specific adenosinedeaminase or catalytic domain thereof.
 69. The composition of claim 68,wherein the RNA-specific adenosine deaminase is ADAR.
 70. Thecomposition of claim 69, wherein the ADAR is human ADAR (huADAR) and/orADAR1 or ADAR2, or a catalytic domain thereof.
 71. The composition ofclaim 70, wherein the ADAR is huADAR or a catalytic domain thereof. 72.The composition of claim 69, wherein the ADAR is a mutated hADAR2dcomprising mutation E488Q or a mutated hADAR1d comprising mutationE1008Q.
 73. The composition of claim 51, wherein the Cas13b effectorprotein comprises one or more heterologous nuclear export signals (NES)or nuclear localization signals (NLS).
 74. The composition of claim 73,wherein the NES is an HIV Rev NES or MAPK NES.
 75. The composition ofclaim 73, wherein the heterologous NES or NLS is fused at the C-terminalof the Cas13b effector protein.
 76. The composition of claim 51, whereinthe target RNA sequence is within a cell.
 77. A cell comprising thecomposition of claim
 51. 78. The cell of claim 77, wherein the cell is aprokaryotic cell.
 79. The cell of claim 77, wherein the cell is aeukaryotic cell.
 80. The eukaryotic cell of claim 79, wherein the cellis a mammalian cell, a human cell, a non-human animal cell, or a plantcell.
 81. A cell line comprising the cell of claim 79, or progenythereof.
 82. A non-human animal comprising the cell of any one of claim79.
 83. A plant comprising the cell of claim
 79. 84. A method ofprophylactically or therapeutically treating a subject in need thereof,comprising the composition of claim
 51. 85. A method of prophylacticallyor therapeutically treating a subject in need thereof, comprising thecell of claim
 79. 86. A method of modifying a target RNA sequence, saidmethod comprising delivering to said target RNA sequence a compositioncomprising: (a) a Cas13b effector protein comprising any one of aminoacid sequences of SEQ ID NO: 31-38, (b) an engineered guide molecule,and (c) one or more heterologous functional domains associated with theCas13b effector protein, wherein the engineered guide molecule iscapable of forming a CRISPR-Cas complex with the Cas13b effector proteinand comprises (i) a guide sequence that directs sequence-specificbinding to a target RNA sequence other than the naturally occurringCas13b protospacer and that reprograms the CRISPR-Cas complex to bindsaid target RNA sequence, and (b) a direct repeat sequence, wherein theone or more heterologous functional domains modifies the target RNAsequence, and wherein the target RNA sequence is in a prokaryotic cellor a eukaryotic cell.
 87. The method of claim 86, wherein the one ormore heterologous functional domains modifies the target RNA sequence byconverting adenosine to inosine.
 88. The method of claim 87, wherein theone or more heterologous functional domains comprises an adenosinedeaminase.
 89. The method of claim 88, wherein the Cas13b effectorprotein, engineered guide molecule, and a coding sequence of theadenosine deaminase, are delivered as one or more polynucleotidemolecules or as a ribonucleoprotein complex.
 90. The method of claim 89,wherein the Cas13b effector protein, engineered guide molecule, andcoding sequence of the adenosine deaminase are delivered by means ofparticles, vesicles, or one or more viral vectors.